Antisense modulation of p38 mitogen activated protein kinase expression

ABSTRACT

Compositions and methods for the treatment and diagnosis of diseases or conditions amenable to treatment through modulation of expression of a gene encoding a p38 mitogen-activated protein kinase (p38 MAPK) are provided. Methods for the treatment and diagnosis of diseases or conditions associated with aberrant expression of one or more p38 MAPKs are also provided.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 10/238,442, filed Sep. 9, 2002, which is acontinuation of U.S. patent application Ser. No. 09/640,101 filed Aug.15, 2000, now issued as U.S. Pat. No. 6,448,079, which is acontinuation-in-part of U.S. patent application Ser. No. 09/286,904,filed Apr. 6, 1999, now issued as U.S. Pat. No. 6,140,124.

FIELD OF THE INVENTION

[0002] This invention relates to compositions and methods for modulatingexpression of p38 mitogen activated protein kinase genes, a family ofnaturally present cellular genes involved in signal transduction, andinflammatory and apoptotic responses. This invention is also directed tomethods for inhibiting inflammation or apoptosis; these methods can beused diagnostically or therapeutically. Furthermore, this invention isdirected to treatment of diseases or conditions associated withexpression of p38 mitogen activated protein kinase genes.

BACKGROUND OF THE INVENTION

[0003] Cellular responses to external factors, such as growth factors,cytokines, and stress conditions, result in altered gene expression.These signals are transmitted from the cell surface to the nucleus bysignal transduction pathways. Beginning with an external factor bindingto an appropriate receptor, a cascade of signal transduction events isinitiated. These responses are mediated through activation of variousenzymes and the subsequent activation of specific transcription factors.These activated transcription factors then modulate the expression ofspecific genes.

[0004] The phosphorylation of enzymes plays a key role in thetransduction of extracellular signals into the cell. Mitogen activatedprotein kinases (MAPKs), enzymes which effect such phosphorylations aretargets for the action of growth factors, hormones, and other agentsinvolved in cellular metabolism, proliferation and differentiation (Cobbet al., J. Biol. Chem., 1995, 270, 14843). Mitogen activated proteinkinases were initially discovered due to their ability to be tyrosinephosphorylated in response to exposure to bacterial lipopolysaccharidesor hyperosmotic conditons (Han et al, Science, 1994, 265, 808). Theseconditions activate inflammatory and apoptotic responses mediated byMAPK. In general, MAP kinases are involved in a variety of signaltransduction pathways (sometimes overlapping and sometimes parallel)that function to convey extracellular stimuli to protooncogene productsto modulate cellular proliferation and/or differentiation (Seger et al.,FASEB J., 1995, 9, 726; Cano et al., Trends Biochem. Sci., 1995, 20,117).

[0005] One of the MAPK signal transduction pathways involves the MAPkinases p38α and p38β (also known as CSaids Binding Proteins, CSBP).These MAP kinases are responsible for the phosphorylation of ATF-2,MEFC2 and a variety of other cellular effectors that may serve assubstrates for p38 MAPK proteins (Kummer et al, J. Biol. Chem., 1997,272, 20490). Phosphorylation of p38 MAPKs potentiates the ability ofthese factors to activate transcription (Raingeaud et al, Mol. CellBio., 1996, 16, 1247; Han et al, Nature, 1997, 386, 296). Among thegenes activated by the p38 MAPK signaling pathway is IL-6 (De Cesaris,P., et al., J. Biol. Chem., 1998, 273, 7566-7571).

[0006] Besides p38α and p38β, other p38 MAPK family members have beendescribed, including p38γ (Li et al, Biochem. Biophys. Res. Commun.,1996, 228, 334), and p38δ (Jiang et al, J. Biol. Chem., 1997, 272,30122). The term “p38” as used herein shall mean a member of the p38MAPK family, including but not limited to p38α, p38β, p38γ and p38δ,their isoforms (Kumar et al, Biochem. Biophys. Res. Commun., 1997, 235,533) and other members of the p38 MAPK family of proteins whether theyfunction as p38 MAP kinases per se or not.

[0007] Modulation of the expression of one or more p38 MAPKs isdesirable in order to interfere with inflammatory or apoptotic responsesassociated with disease states and to modulate the transcription ofgenes stimulated by ATF-2, MEFC2 and other p38 MAPK phosphorylationsubstrates.

[0008] Inhibitors of p38 MAPKs have been shown to have efficacy inanimal models of arthritis (Badger, A. M., et al., J. Pharmacol. Exp.Ther., 1996, 279, 1453-1461) and angiogenesis (Jackson, J. R., et al.,J. Pharmacol. Exp. Ther., 1998, 284, 687-692). MacKay, K. andMochy-Rosen, D. (J. Biol. Chem., 1999, 274, 6272-6279) demonstrate thatan inhibitor of p38 MAPKs prevents apoptosis during ischemia in cardiacmyocytes, suggesting that p38 MAPK inhibitors can be used for treatingischemic heart disease. p38 MAPK also is required for T-cell HIV-1replication (Cohen et al, Mol. Med., 1997, 3, 339) and may be a usefultarget for AIDS therapy. Other diseases believed to be amenable totreatment by inhibitors of p38 MAPKs are disclosed in U.S. Pat. No.5,559,137, herein incorporated by reference.

[0009] Therapeutic agents designed to target p38 MAPKs include smallmolecule inhibitors and antisense oligonucleotides. Small moleculeinhibitors based on pyridinyl imidazole are described in U.S. Pat. Nos.5,670,527; 5,658,903; 5,656,644; 5,559,137; 5,593,992; and 5,593,991. WO98/27098 and WO 99/00357 describe additional small molecule inhibitors,one of which has entered clinical trials. Other small moleculeinhibitors are also known.

[0010] Antisense therapy represents a potentially more specific therapyfor targeting p38 MAPKs and, in particular, specific p38 MAPK isoforms.Nagata, Y., et al. (Blood, 1998, 6, 1859-1869) disclose an antisensephosphothioester oligonucleotide targeted to the translational startsite of mouse p38b (p38β). Aoshiba, K., et al. (J. Immunol., 1999, 162,1692-1700) and Cohen, P. S., et al. (Mol. Med., 1997, 3, 339-346)disclose a phosphorothioate antisense oligonucleotide targeted to thecoding regions of human p38α, human p38β and rat p38.

[0011] There remains a long-felt need for improved compositions andmethods for modulating the expression of p38 MAP kinases.

SUMMARY OF THE INVENTION

[0012] The present invention provides antisense compounds which aretargeted to nucleic acids encoding a p38 MAPK and are capable ofmodulating p38 MAPK expression. The present invention also providesoligonucleotides targeted to nucleic acids encoding a p38 MAPK. Thepresent invention also comprises methods of modulating the expression ofa p38 MAPK, in cells and tissues, using the oligonucleotides of theinvention. Methods of inhibiting p38 MAPK expression are provided; thesemethods are believed to be useful both therapeutically anddiagnostically. These methods are also useful as tools, for example, fordetecting and determining the role of p38 MAPKs in various cellfunctions and physiological processes and conditions and for diagnosingconditions associated with expression of p38 MAPKs.

[0013] The present invention also comprises methods for diagnosing andtreating inflammatory diseases, particularly rheumatoid arthritis andasthma. These methods are believed to be useful, for example, indiagnosing p38 MAPK-associated disease progression. These methods employthe oligonucleotides of the invention. These methods are believed to beuseful both therapeutically, including prophylactically, and as clinicalresearch and diagnostic tools.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIGS. 1A-1B are graphs showing the effect of inhaled p38α MAPkinase antisense oligonucleotide ISIS 101757 (ASO, FIG. 1A) andmismatched control oligonucleotide ISIS 101758 (MM ASO, FIG. 1B) onovalbumin (OVA)-induced airway hyperresponsiveness in a murine asthmamodel.

[0015]FIG. 2 is a graph showing that inhaled ISIS 101757 increases theprovocation concentration of methacholine required to achieve doublingof airway reactivity (PC200) in OVA-challenged mice.

[0016]FIGS. 3A-3B are graphs showing the effect of inhaled ISIS 101757(FIG. 3A) and 101758 (FIG. 3B) on immune cells in broncheolar lavage(BAL) fluid of OVA-challenged mice. EOS=eosinpophils, NEU=neutrophils,MAC=macrophages, LYM=lymphocyes.

[0017]FIG. 4 is a graph showing aerosolized ISIS 101757 concentration inmouse lung vs. dose.

[0018]FIG. 5 is a graph showing dose-dependent inhibition of the penhresponse to methacholine (50 mg/ml) challenge by ISIS 101757. ISIS101757 doses are in mg/kg α-axis).

[0019]FIG. 6 is a graph showing ISIS 101757 concentration (μg/g) in thelungs vs. dose (intratracheal administration).

DETAILED DESCRIPTION OF THE INVENTION

[0020] p38 MAPKs play an important role in signal transduction inresponse to cytokines, growth factors and other cellular stimuli.Specific responses elicited by p38 include inflammatory and apoptoticresponses. Modulation of p38 may be useful in the treatment ofinflammatory diseases, such as rheumatoid arthritis.

[0021] The present invention employs antisense compounds, particularlyoligonucleotides, for use in modulating the function of nucleic acidmolecules encoding a p38 MAPK, ultimately modulating the amount of a p38MAPK produced. This is accomplished by providing oligonucleotides whichspecifically hybridize with nucleic acids, preferably mRNA, encoding ap38 MAPK.

[0022] The antisense compounds may be used to modulate the function of aparticular p38 MAPK isoform, e.g. for research purposes to determine therole of a particular isoform in a normal or disease process, or to treata disease or condition that may be associated with a particular isoform.It may also be desirable to target multiple p38 MAPK isoforms. In eachcase, antisense compounds can be designed by taking advantage ofsequence homology between the various isoforms. If an antisense compoundto a particular isoform is desired, then the antisense compound isdesigned to a unique region in the desired isoform's gene sequence. Withsuch a compound, it is desirable that this compound does not inhibit theexpression of other isoforms. Less desirable, but acceptable, arecompounds that do not “substantially” inhibit other isoforms. By“substantially”, it is intended that these compounds do not inhibit theexpression of other isoforms greater than 25%; more preferred arecompounds that do not inhibit other isoforms greater than 10%. If anantisense compound is desired to target multiple p38 isoforms, thenregions of significant homology between the isoforms can be used.

[0023] This relationship between an antisense compound such as anoligonucleotide and its complementary nucleic acid target, to which ithybridizes, is commonly referred to as “antisense”. “Targeting” anoligonucleotide to a chosen nucleic acid target, in the context of thisinvention, is a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid from an infectious agent. In the present invention,the target is a nucleic acid encoding a p38 MAPK; in other words, a p38MAPK gene or RNA expressed from a p38 MAPK gene. p38 MAPK mRNA ispresently the preferred target. The targeting process also includesdetermination of a site or sites within the nucleic acid sequence forthe antisense interaction to occur such that modulation of geneexpression will result.

[0024] In accordance with this invention, persons of ordinary skill inthe art will understand that messenger RNA includes not only theinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form a region known to suchpersons as the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. Thus,oligonucleotides may be formulated in accordance with this inventionwhich are targeted wholly or in part to these associated ribonucleotidesas well as to the informational ribonucleotides. The oligonucleotide maytherefore be specifically hybridizable with a transcription initiationsite region, a translation initiation codon region, a 5′ cap region, anintron/exon junction, coding sequences, a translation termination codonregion or sequences in the 5′- or 3′-untranslated region. Since, as isknown in the art, the translation initiation codon is typically 5′-AUG(in transcribed mRNA molecules; 5′-ATG in the corresponding DNAmolecule), the translation initiation codon is also referred to as the“AUG codon,” the “start codon” or the “AUG start codon.” A minority ofgenes have a translation initiation codon having the RNA sequence5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shownto function in vivo. Thus, the terms “translation initiation codon” and“start codon” can encompass many codon sequences, even though theinitiator amino acid in each instance is typically methionine (ineukaryotes) or formylmethionine (prokaryotes). It is also known in theart that eukaryotic and prokaryotic genes may have two or morealternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAmolecule transcribed from a gene encoding p38, regardless of thesequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. This region is a preferred targetregion. Similarly, the terms “stop codon region” and “translationtermination codon region” refer to a portion of such an mRNA or genethat encompasses from about 25 to about 50 contiguous nucleotides ineither direction (i.e., 5′ or 3′) from a translation termination codon.This region is a preferred target region. The open reading frame (ORF)or “coding region,” which is known in the art to refer to the regionbetween the translation initiation codon and the translation terminationcodon, is also a region which may be targeted effectively. Otherpreferred target regions include the 5′ untranslated region (5′UTR),known in the art to refer to the portion of an mRNA in the 5′ directionfrom the translation initiation codon, and thus including nucleotidesbetween the 5′ cap site and the translation initiation codon of an mRNAor corresponding nucleotides on the gene) and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA or corresponding nucleotides on the gene). mRNA splice sites mayalso be preferred target regions, and are particularly useful insituations where aberrant splicing is implicated in disease, or where anoverproduction of a particular mRNA splice product is implicated indisease. Aberrant fusion junctions due to rearrangements or deletionsmay also be preferred targets.

[0025] Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired modulation.

[0026] “Hybridization”, in the context of this invention, means hydrogenbonding, also known as Watson-Crick base pairing, between complementarybases, usually on opposite nucleic acid strands or two regions of anucleic acid strand. Guanine and cytosine are examples of complementarybases which are known to form three hydrogen bonds between them. Adenineand thymine are examples of complementary bases which form two hydrogenbonds between them.

[0027] “Specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity such thatstable and specific binding occurs between the DNA or RNA target and theoligonucleotide.

[0028] It is understood that an oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment and, in the case of invitro assays, under conditions in which the assays are conducted.

[0029] Hybridization of antisense oligonucleotides with mRNA interfereswith one or more of the normal functions of mRNA. The functions of mRNAto be interfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.

[0030] The overall effect of interference with mRNA function ismodulation of p38 MAPK expression. In the context of this invention“modulation” means either inhibition or stimulation; i.e., either adecrease or increase in expression. This modulation can be measured inways which are routine in the art, for example by Northern blot assay ofmRNA expression as taught in the examples of the instant application orby Western blot or ELISA assay of protein expression, or by animmunoprecipitation assay of protein expression, as taught in theexamples of the instant application. Effects on cell proliferation ortumor cell growth can also be measured, as taught in the examples of theinstant application.

[0031] The oligonucleotides of this invention can be used indiagnostics, therapeutics, prophylaxis, and as research reagents and inkits. Since the oligonucleotides of this invention hybridize to nucleicacids encoding a p38 MAPK, sandwich, calorimetric and other assays caneasily be constructed to exploit this fact. Furthermore, since theoligonucleotides of this invention hybridize specifically to nucleicacids encoding particular isoforms of p38 MAPK, such assays can bedevised for screening of cells and tissues for particular p38 MAPKisoforms. Such assays can be utilized for diagnosis of diseasesassociated with various p38 MAPK isoforms. Provision of means fordetecting hybridization of oligonucleotide with a p38 MAPK gene or mRNAcan routinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of p38 MAPK may also beprepared.

[0032] The present invention is also suitable for diagnosing abnormalinflammatory states in tissue or other samples from patients suspectedof having an inflammatory disease such as rheumatoid arthritis. Theability of the oligonucleotides of the present invention to inhibitinflammation may be employed to diagnose such states. A number of assaysmay be formulated employing the present invention, which assays willcommonly comprise contacting a tissue sample with an oligonucleotide ofthe invention under conditions selected to permit detection and,usually, quantitation of such inhibition. In the context of thisinvention, to “contact” tissues or cells with an oligonucleotide oroligonucleotides means to add the oligonucleotide(s), usually in aliquid carrier, to a cell suspension or tissue sample, either in vitroor ex vivo, or to administer the oligonucleotide(s) to cells or tissueswithin an animal. Similarly, the present invention can be used todistinguish p38 MAPK-associated diseases, from diseases having otheretiologies, in order that an efficacious treatment regime can bedesigned.

[0033] The oligonucleotides of this invention may also be used forresearch purposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

[0034] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleicacid. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intersugar(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced binding to target and increased stability in thepresence of nucleases.

[0035] The antisense compounds in accordance with this inventionpreferably comprise from about 5 to about 50 nucleobases. Particularlypreferred are antisense oligonucleotides comprising from about 8 toabout 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides).Preferred embodiments comprise at least an 8-nucleobase portion of asequence of an antisense compound which inhibits the expression of a p38mitogen activated kinase. As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2=, 3=or 5=hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn the respective ends of thislinear polymeric structure can be further joined to form a circularstructure, however, open linear structures are generally preferred.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3=to5=phosphodiester linkage.

[0036] While the preferred form of antisense compound is asingle-stranded antisense oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

[0037] The first evidence that dsRNA could lead to gene silencing inanimals came in 1995 from work in the nematode, Caenorhabditis elegans(Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. haveshown that the primary interference effects of dsRNA areposttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,1998, 95, 15502-15507). The posttranscriptional antisense mechanismdefined in Caenorhabditis elegans resulting from exposure todouble-stranded RNA (dsRNA) has since been designated RNA interference(RNAi). This term has been generalized to mean antisense-mediated genesilencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). Recently, it has been shown that itis, in fact, the single-stranded RNA oligomers of antisense polarity ofthe dsRNAs which are the potent inducers of RNAi (Tijsterman et al.,Science, 2002, 295, 694-697). Single stranded and double stranded RNA(RNAi) inhibition of human p38 MAP kinase is also within the scope ofthe present invention.

[0038] Oligomer and Monomer Modifications

[0039] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside linkage or in conjunctionwith the sugar ring the backbone of the oligonucleotide. The normalinternucleoside linkage that makes up the backbone of RNA and DNA is a3′ to 5′ phosphodiester linkage.

[0040] Modified Internucleoside Linkages

[0041] Specific examples of preferred antisense oligomeric compoundsuseful in this invention include oligonucleotides containing modifiede.g. non-naturally occurring internucleoside linkages. As defined inthis specification, oligonucleotides having modified internucleosidelinkages include internucleoside linkages that retain a phosphorus atomand internucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

[0042] In the C. elegans system, modification of the internucleotidelinkage (phosphorothioate) did not significantly interfere with RNAiactivity. Based on this observation, it is suggested that certainpreferred oligomeric compounds of the invention can also have one ormore modified internucleoside linkages. A preferred phosphoruscontaining modified internucleoside linkage is the phosphorothioateinternucleoside linkage.

[0043] Preferred modified oligonucleotide backbones containing aphosphorus atom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphoro-dithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0044] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0045] In more preferred embodiments of the invention, oligomericcompounds have one or more phosphorothioate and/or heteroatominternucleoside linkages, in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester internucleotide linkage isrepresented as —O—P(═O)(OH)—O—CH₂—]. The MMI type internucleosidelinkages are disclosed in the above referenced U.S. Pat. No. 5,489,677.Preferred amide internucleoside linkages are disclosed in the abovereferenced U.S. Pat. No. 5,602,240.

[0046] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0047] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0048] Oligomer Mimetics

[0049] Another preferred group of oligomeric compounds amenable to thepresent invention includes oligonucleotide mimetics. The term mimetic asit is applied to oligonucleotides is intended to include oligomericcompounds wherein only the furanose ring or both the furanose ring andthe internucleotide linkage are replaced with novel groups, replacementof only the furanose ring is also referred to in the art as being asugar surrogate. The heterocyclic base moiety or a modified heterocyclicbase moiety is maintained for hybridization with an appropriate targetnucleic acid. One such oligomeric compound, an oligonucleotide mimeticthat has been shown to have excellent hybridization properties, isreferred to as a peptide nucleic acid (PNA). In PNA oligomericcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA oligomericcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA oligomeric compounds can be found inNielsen et al., Science, 1991, 254, 1497-1500.

[0050] One oligonucleotide mimetic that has been reported to haveexcellent hybridization properties is peptide nucleic acids (PNA). Thebackbone in PNA compounds is two or more linked aminoethylglycine unitswhich gives PNA an amide containing backbone. The heterocyclic basemoieties are bound directly or indirectly to aza nitrogen atoms of theamide portion of the backbone. Representative United States patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which isherein incorporated by reference. Further teaching of PNA compounds canbe found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0051] PNA has been modified to incorporate numerous modifications sincethe basic PNA structure was first prepared. The basic structure is shownbelow:

[0052] wherein

[0053] Bx is a heterocyclic base moiety;

[0054] T₄ is hydrogen, an amino protecting group, —C(O)R₅, substitutedor unsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

[0055] T₅ is —OH, —N(Z₁) Z₂, R₅, D or L α-amino acid linked via theα-amino group or optionally through the ω-amino group when the aminoacid is lysine or ornithine or a peptide derived from D, L or mixed Dand L amino acids linked through an amino group, a chemical functionalgroup, a reporter group or a conjugate group;

[0056] Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

[0057] Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)_(n)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via theα-carboxyl group or optionally through the ω-carboxyl group when theamino acid is aspartic acid or glutamic acid or a peptide derived fromD, L or mixed D and L amino acids linked through a carboxyl group;

[0058] Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl,—C(═O)—CH₃, benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

[0059] each J is O, S or NH;

[0060] R₅ is a carbonyl protecting group; and

[0061] n is from 2 to about 50.

[0062] Another class of oligonucleotide mimetic that has been studied isbased on linked morpholino units (morpholino nucleic acid) havingheterocyclic bases attached to the morpholino ring. A number of linkinggroups have been reported that link the morpholino monomeric units in amorpholino nucleic acid. A preferred class of linking groups have beenselected to give a non-ionic oligomeric compound. The non-ionicmorpholino-based oligomeric compounds are less likely to have undesiredinteractions with cellular proteins. Morpholino-based oligomericcompounds are non-ionic mimics of oligonucleotides which are less likelyto form undesired interactions with cellular proteins (Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41 (14), 4503-4510).Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No.5,034,506, issued Jul. 23, 1991. The morpholino class of oligomericcompounds have been prepared having a variety of different linkinggroups joining the monomeric subunits.

[0063] Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

[0064] wherein

[0065] T₁ is hydroxyl or a protected hydroxyl;

[0066] T₅ is hydrogen or a phosphate or phosphate derivative;

[0067] L₂ is a linking group; and

[0068] n is from 2 to about 50.

[0069] A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichrdism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

[0070] The general formula of CeNA is shown below:

[0071] wherein

[0072] each Bx is a heterocyclic base moiety;

[0073] T₁ is hydroxyl or a protected hydroxyl; and

[0074] T2 is hydroxyl or a protected hydroxyl.

[0075] Another class of oligonucleotide mimetic (anhydrohexitol nucleicacid) can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

[0076] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom ofthe sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage therebyforming a bicyclic sugar moiety. The linkage is preferably a methylene(—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNAand LNA analogs display very high duplex thermal stabilities withcomplementary DNA and RNA (Tm=+3 to +10 C), stability towards3′-exonucleolytic degradation and good solubility properties. The basicstructure of LNA showing the bicyclic ring system is shown below:

[0077] The conformations of LNAs determined by 2D NMR spectroscopy haveshown that the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

[0078] LNA has been shown to form exceedingly stable LNA:LNA duplexes(Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

[0079] LNAs also form duplexes with complementary DNA, RNA or LNA withhigh thermal affinities. Circular dichroism (CD) spectra show thatduplexes involving fully modified LNA (esp. LNA:RNA) structurallyresemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR)examination of an LNA:DNA duplex confirmed the 3′-endo conformation ofan LNA monomer. Recognition of double-stranded DNA has also beendemonstrated suggesting strand invasion by LNA. Studies of mismatchedsequences show that LNAs obey the Watson-Crick base pairing rules withgenerally improved selectivity compared to the corresponding unmodifiedreference strands.

[0080] Novel types of LNA-oligomeric compounds, as well as the LNAs, areuseful in a wide range of diagnostic and therapeutic applications. Amongthese are antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs. Potent and nontoxic antisense oligonucleotidescontaining LNAs have been described (Wahlestedt et al., Proc. Natl.Acad. Sci. U.S. A., 2000, 97, 5633-5638.) The authors have demonstratedthat LNAs confer several desired properties to antisense agents. LNA/DNAcopolymers were not degraded readily in blood serum and cell extracts.LNA/DNA copolymers exhibited potent antisense activity in assay systemsas disparate as G-protein-coupled receptor signaling in living rat brainand detection of reporter genes in Escherichia coli. Lipofectin-mediatedefficient delivery of LNA into living human breast cancer cells has alsobeen accomplished.

[0081] The synthesis and preparation of the LNA monomers adenine,cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along withtheir oligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

[0082] The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs,have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998,8, 2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2‘-methylamino-LNA’s have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

[0083] Further oligonucleotide mimetics have been prepared to includebicyclic and tricyclic nucleoside analogs having the formulas (amiditemonomers shown):

[0084] (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439;Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberget al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These modifiednucleoside analogs have been oligomerized using the phosphoramiditeapproach and the resulting oligomeric compounds containing tricyclicnucleoside analogs have shown increased thermal stabilities (Tm's) whenhybridized to DNA, RNA and itself. Oligomeric compounds containingbicyclic nucleoside analogs have shown thermal stabilities approachingthat of DNA duplexes.

[0085] Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

[0086] The general formula (for definitions of Markush variables see:U.S. Pat. Nos. 5,874,553 and 6,127,346 herein incorporated by referencein their entirety) is shown below.

[0087] Another oligonucleotide mimetic has been reported wherein thefuranosyl ring has been replaced by a cyclobutyl moiety.

[0088] Modified Sugars

[0089] Oligomeric compounds of the invention may also contain one ormore substituted sugar moieties. Preferred oligomeric compounds comprisea sugar substituent group selected from: OH; F; O—, S—, or N-alkyl; O—,S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise a sugarsubstituent group selected from: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-C—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

[0090] Other preferred sugar substituent groups include methoxy(—O—CH₃), aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl(—O—CH₂—CH═CH₂) and fluoro (F). 2′-Sugar substituent groups may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligomeric compound, particularly the 3′position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligomeric compounds may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0091] Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

[0092] wherein:

[0093] R_(b) is O, S or NH;

[0094] R_(d) is a single bond, O, S or C(═O);

[0095] R_(e) is C₁-C₁₀ alkyl, N(R_(k)) (R_(m)), N(R_(k)) (R_(n)),N═C(R_(p)) (R_(q)), N═C(R_(p)) (R_(r)) or has formula III_(a);

[0096] R_(p) and R_(q) are each independently hydrogen or C₁-C₁₀ alkyl;

[0097] R_(r) is —R_(x)—R_(y);

[0098] each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

[0099] or optionally, R_(u) and R_(v), together form a phthalimidomoiety with the nitrogen atom to which they are attached;

[0100] each R_(w) is, independently, substituted or unsubstituted C₁-C₁₀alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy,allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

[0101] R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0102] R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

[0103] R_(x) is a bond or a linking moiety;

[0104] R_(y) is a chemical functional group, a conjugate group or asolid support medium;

[0105] each R_(m) and R_(n) is, independently, H, a nitrogen protectinggroup, substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, wherein the substituent groups are selected from hydroxyl,amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy,halogen, alkyl, aryl, alkenyl, alkynyl; NH₃ ⁺, N(R_(u)) (R_(v)),guanidino and acyl where said acyl is an acid amide or an ester;

[0106] or R_(m) and R_(n), together, are a nitrogen protecting group,are joined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

[0107] R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

[0108] each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

[0109] R_(f), R_(g) and R_(h) comprise a ring system having from about 4to about 7 carbon atoms or having from about 3 to about 6 carbon atomsand 1 or 2 heteroatoms wherein said heteroatoms are selected fromoxygen, nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

[0110] R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms,alkenyl having 2 to about 10 carbon atoms, alkynyl having 2 to about 10carbon atoms, aryl having 6 to about 14 carbon atoms, N(R_(k)) (R_(m))OR_(k), halo, SR_(k) or CN;

[0111] m_(a) is 1 to about 10;

[0112] each mb is, independently, 0 or 1;

[0113] mc is 0 or an integer from 1 to 10;

[0114] md is an integer from 1 to 10;

[0115] me is from 0, 1 or 2; and

[0116] provided that when mc is 0, md is greater than 1.

[0117] Representative substituents groups of Formula I are disclosed inUnited States patent application Ser. No. 09/130,973, filed Aug. 7,1998, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” herebyincorporated by reference in its entirety.

[0118] Representative cyclic substituent groups of Formula II aredisclosed in U.S. patent application Ser. No. 09/123,108, filed Jul. 27,1998, entitled “RNA Targeted 2′-Oligomeric compounds that areConformationally Preorganized,” hereby incorporated by reference in itsentirety.

[0119] Particularly preferred sugar substituent groups includeO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10.

[0120] Representative guanidino substituent groups that are shown informula III and IV are disclosed in co-owned U.S. patent applicationSer. No. 09/349,040, entitled “Functionalized Oligomers”, filed Jul. 7,1999, hereby incorporated by reference in its entirety.

[0121] Representative acetamido substituent groups are disclosed in U.S.Pat. No. 6,147,200 which is hereby incorporated by reference in itsentirety.

[0122] Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

[0123] Modified Nucleobases/Naturally Occurring Nucleobases

[0124] Oligomeric compounds may also include nucleobase (often referredto in the art simply as “base” or “heterocyclic base moiety”)modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases also referred herein as heterocyclic base moietiesinclude other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0125] Heterocyclic base moieties may also include those in which thepurine or pyrimidine base is replaced with other heterocycles, forexample 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in The Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

[0126] In one aspect of the present invention oligomeric compounds areprepared having polycyclic heterocyclic compounds in place of one ormore heterocyclic base moieties. A number of tricyclic heterocycliccompounds have been previously reported. These compounds are routinelyused in antisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

[0127] Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁—R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁—R₁₄═H), [Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁—R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.Patent Application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. Patent Application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

[0128] Further helix-stabilizing properties have been observed when acytosine analog/substitute has an aminoethoxy moiety attached to therigid 1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁═—O—(CH₂)₂—NH₂,R₁₂₋₁₄═H) [Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120,8531-8532]. Binding studies demonstrated that a single incorporationcould enhance the binding affinity of a model oligonucleotide to itscomplementary target DNA or RNA with a ΔT_(m) of up to 18° relative to5-methyl cytosine (dC5^(me)), which is the highest known affinityenhancement for a single modification, yet. On the other hand, the gainin helical stability does not compromise the specificity of theoligonucleotides. The T_(m) data indicate an even greater discriminationbetween the perfect match and mismatched sequences compared to dC5^(me).It was suggested that the tethered amino group serves as an additionalhydrogen bond donor to interact with the Hoogsteen face, namely the O6,of a complementary guanine thereby forming 4 hydrogen bonds. This meansthat the increased affinity of G-clamp is mediated by the combination ofextended base stacking and additional specific hydrogen bonding.

[0129] Further tricyclic heterocyclic compounds and methods of usingthem that are amenable to the present invention are disclosed in U.S.Pat. Ser. No. 6,028,183, which issued on May 22, 2000, and U.S. Pat.Ser. No. 6,007,992, which issued on Dec. 28, 1999, the contents of bothare commonly assigned with this application and are incorporated hereinin their entirety.

[0130] The enhanced binding affinity of the phenoxazine derivativestogether with their uncompromised sequence specificity makes themvaluable nucleobase analogs for the development of more potentantisense-based drugs. In fact, promising data have been derived from invitro experiments demonstrating that heptanucleotides containingphenoxazine substitutions are capable to activate RNaseH, enhancecellular uptake and exhibit an increased antisense activity [Lin, K-Y;Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532]. The activityenhancement was even more pronounced in case of G-clamp, as a singlesubstitution was shown to significantly improve the in vitro potency ofa 20mer 2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.;Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci,M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, tooptimize oligonucleotide design and to better understand the impact ofthese heterocyclic modifications on the biological activity, it isimportant to evaluate their effect on the nuclease stability of theoligomers.

[0131] Further modified polycyclic heterocyclic compounds useful asheterocyclcic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare commonly owned with the instant application, and each of which isherein incorporated by reference.

[0132] The oligonucleotides of the present invention also includevariants in which a different base is present at one or more of thenucleotide positions in the oligonucleotide. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the oligonucleotide. Thus, a 20-mer may comprise60 variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression of HCVmRNA and/or HCV replication.

[0133] Conjugates

[0134] A further preferred substitution that can be appended to theoligomeric compounds of the invention involves the linkage of one ormore moieties or conjugates which enhance the activity, cellulardistribution or cellular uptake of the resulting oligomeric compounds.In one embodiment such modified oligomeric compounds are prepared bycovalently attaching conjugate groups to functional groups such ashydroxyl or amino groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugates groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve oligomer uptake, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA. Groups that enhance the pharmacokineticproperties, in the context of this invention, include groups thatimprove oligomer uptake, distribution, metabolism or excretion.Representative conjugate groups are disclosed in International PatentApplication PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure ofwhich is incorporated herein by reference. Conjugate moieties includebut are not limited to lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-o-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

[0135] The oligomeric compounds of the invention may also be conjugatedto active drug substances, for example, aspirin, warfarin,phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in United States patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0136] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0137] Chimeric Oligomeric Compounds

[0138] It is not necessary for all positions in an oligomeric compoundto be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single oligomericcompound or even at a single monomeric subunit such as a nucleosidewithin a oligomeric compound. The present invention also includesoligomeric compounds which are chimeric oligomeric compounds. “Chimeric”oligomeric compounds or “chimeras,” in the context of this invention,are oligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

[0139] Chimeric oligomeric compounds typically contain at least oneregion modified so as to confer increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligomeric compound may serve as a substrate for enzymes capable ofcleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligomeric compounds when chimeras are used, compared to forexample phosphorothioate deoxyoligonucleotides hybridizing to the sametarget region. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

[0140] Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797;5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350;5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference in its entirety.

[0141] 3′-Endo Modifications

[0142] In one aspect of the present invention oligomeric compoundsinclude nucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

[0143] Nucleoside conformation is influenced by various factorsincluding substitution at the 2′, 3′ or 4′-positions of thepentofuranosyl sugar. Electronegative substituents generally prefer theaxial positions, while sterically demanding substituents generallyprefer the equatorial positions (Principles of Nucleic Acid Structure,Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ positionto favor the 3′-endo conformation can be achieved while maintaining the2′-OH as a recognition element, as illustrated in FIG. 2, below (Galloet al., Tetrahedronl (2001), 57, 5707-5713. Harry- O'kuru et al., J.Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem(1999), 64, 747-754.) Alternatively, preference for the 3′-endoconformation can be achieved by deletion of the 2′-OH as exemplified by2′deoxy-2′ F-nucleosides (Kawasaki et al., J. Med. Chel . (1993), 36,831-841), which adopts the 3′-endo conformation positioning theelectronegative fluorine atom in the axial position. Other modificationsof the ribose ring, for example substitution at the 4′-position to give4′-F modified nucleosides (Guillerm et al., Bioorganic and MedicinalChemistry Lettersl (1995), 5, 1455-1460 and Owen et al., J. Org. Chem .(1976), 41, 3010-3017), or for example modification to yieldmethanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett .(2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal ChemistryLetters (2001), 11, 1333-1337) also induce preference for the 3′-endoconformation. Along similar lines, oligomeric triggers of RNAi responsemight be composed of one or more nucleosides modified in such a way thatconformation is locked into a C3′-endo type conformation, i.e. LockedNucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), andethylene bridged Nucleic Acids (ENA, Morita et al, Bioorganic &Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of modifiednucleosides amenable to the present invention are shown below in TableI. These examples are meant to be representative and not exhaustive.TABLE I

[0144] The preferred conformation of modified nucleosides and theiroligomers can be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligoncleotides of the presentinvention. The synthesis of numerous modified nucleosides amenable tothe present invention are known in the art (see for example, Chemistryof Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988,Plenum press., and the examples section below.)

[0145] In one aspect, the present invention is directed tooligonucleotides that are prepared having enhanced properties comparedto native RNA against nucleic acid targets. A target is identified andan oligonucleotide is selected having an effective length and sequencethat is complementary to a portion of the target sequence. Eachnucleoside of the selected sequence is scrutinized for possibleenhancing modifications. A preferred modification would be thereplacement of one or more RNA nucleosides with nucleosides that havethe same 3′-endo conformational geometry. Such modifications can enhancechemical and nuclease stability relative to native RNA while at the sametime being much cheaper and easier to synthesize and/or incorporate intoan oligonulceotide. The selected sequence can be further divided intoregions and the nucleosides of each region evaluated for enhancingmodifications that can be the result of a chimeric configuration.Consideration is also given to the 5′ and 3′-termini as there are oftenadvantageous modifications that can be made to one or more of theterminal nucleosides. The oligomeric compounds of the present inventioninclude at least one 5′-modified phosphate group on a single strand oron at least one 5′-position of a double stranded sequence or sequences.Further modifications are also considered such as internucleosidelinkages, conjugate groups, substitute sugars or bases, substitution ofone or more nucleosides with nucleoside mimetics and any othermodification that can enhance the selected sequence for its intendedtarget. The terms used to describe the conformational geometry ofhomoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. Therespective conformational geometry for RNA and DNA duplexes wasdetermined from X-ray diffraction analysis of nucleic acid fibers(Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.) Ingeneral, RNA:RNA duplexes are more stable and have higher meltingtemperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles ofNucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik etal., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic AcidsRes., 1997, 25, 2627-2634). The increased stability of RNA has beenattributed to several structural features, most notably the improvedbase stacking interactions that result from an A-form geometry (Searleet al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., alsodesignated as Northern pucker, which causes the duplex to favor theA-form geometry. In addition, the 2′ hydroxyl groups of RNA can form anetwork of water mediated hydrogen bonds that help stabilize the RNAduplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the otherhand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., alsoknown as Southern pucker, which is thought to impart a less stableB-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and O4′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a O4′-endo pucker contribution.

[0146] DNA:RNA hybrid duplexes, however, are usually less stable thanpure RNA:RNA duplexes, and depending on their sequence may be eithermore or less stable than DNA:DNA duplexes (Searle et al., Nucleic AcidsRes., 1993, 21, 2051-2056). The structure of a hybrid duplex isintermediate between A- and B-form geometries, which may result in poorstacking interactions (Lane et al., Eur. J. Biochem., 1993, 215,297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez etal., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol.,1996, 264, 521-533). The stability of the duplex formed between a targetRNA and a synthetic sequence is central to therapies such as but notlimited to antisense and RNA interference as these mechanisms requirethe binding of a synthetic oligonucleotide strand to an RNA targetstrand. In the case of antisense, effective inhibition of the mRNArequires that the antisense DNA have a very high binding affinity withthe mRNA. Otherwise the desired interaction between the syntheticoligonucleotide strand and target mRNA strand will occur infrequently,resulting in decreased efficacy.

[0147] One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

[0148] As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

[0149] One synthetic 2′-modification that imparts increased nucleaseresistance and a very high binding affinity to nucleotides is the2-methoxyethoxy (2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J.Biol. Chem., 1997, 272, 11944-12000). One of the immediate advantages ofthe 2′-MOE substitution is the improvement in binding affinity, which isgreater than many similar 2′ modifications such as O-methyl, O-propyl,and O-aminopropyl. Oligonucleotides having the 2′-O-methoxyethylsubstituent also have been shown to be antisense inhibitors of geneexpression with promising features for in vivo use (Martin, P., Helv.Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50,168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; andAltmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative toDNA, the oligonucleotides having the 2′-MOE modification displayedimproved RNA affinity and higher nuclease resistance. Chimericoligonucleotides having 2′-MOE substituents in the wing nucleosides andan internal region of deoxy-phosphorothioate nucleotides (also termed agapped oligonucleotide or gapmer) have shown effective reduction in thegrowth of tumors in animal models at low doses. 2′-MOE substitutedoligonucleotides have also shown outstanding promise as antisense agentsin several disease states. One such MOE substituted oligonucleotide ispresently being investigated in clinical trials for the treatment of CMVretinitis.

[0150] Chemistries Defined

[0151] Unless otherwise defined herein, alkyl means C₁-C₁₂, preferablyC₁-C₈, and more preferably C₁-C₆, straight or (where possible) branchedchain aliphatic hydrocarbyl.

[0152] Unless otherwise defined herein, heteroalkyl means C₁-C₁₂,preferably C₁-C₈, and more preferably C₁-C₆, straight or (wherepossible) branched chain aliphatic hydrocarbyl containing at least one,and preferably about 1 to about 3, hetero atoms in the chain, includingthe terminal portion of the chain. Preferred heteroatoms include N, Oand S. Unless otherwise defined herein, cycloalkyl means C₃-C₁₂,preferably C₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

[0153] Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferablyC₂-C₈, and more preferably C₂-C₆ alkenyl, which may be straight or(where possible) branched hydrocarbyl moiety, which contains at leastone carbon-carbon double bond.

[0154] Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferablyC₂-C₈, and more preferably C₂-C₆ alkynyl, which may be straight or(where possible) branched hydrocarbyl moiety, which contains at leastone carbon-carbon triple bond.

[0155] Unless otherwise defined herein, heterocycloalkyl means a ringmoiety containing at least three ring members, at least one of which iscarbon, and of which 1, 2 or three ring members are other than carbon.Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred heterocycloalkyl groupsinclude morpholino, thiomorpholino, piperidinyl, piperazinyl,homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino,pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl,tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl,furanyl, pyranyl, and tetrahydroisothiazolyl.

[0156] Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Preferred aryl rings haveabout 6 to about 20 ring carbons. Especially preferred aryl ringsinclude phenyl, napthyl, anthracenyl, and phenanthrenyl.

[0157] Unless otherwise defined herein, hetaryl means a ring moietycontaining at least one fully unsaturated ring, the ring consisting ofcarbon and non-carbon atoms. Preferably the ring system contains about 1to about 4 rings. Preferably the number of carbon atoms varies from 1 toabout 12, preferably 1 to about 6, and the total number of ring membersvaries from three to about 15, preferably from about 3 to about 8.Preferred ring heteroatoms are N, O and S. Preferred hetaryl moietiesinclude pyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl,pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl,benzothiophenyl, etc.

[0158] Unless otherwise defined herein, where a moiety is defined as acompound moiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryland alkyl), etc., each of the sub-moieties is as defined herein.

[0159] Unless otherwise defined herein, an electron withdrawing group isa group, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

[0160] Unless otherwise defined herein, the terms halogen and halo havetheir ordinary meanings. Preferred halo (halogen) substituents are Cl,Br, and I.

[0161] The aforementioned optional substituents are, unless otherwiseherein defined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc. In all the preceding formulae, the squiggle (˜) indicatesa bond to an oxygen or sulfur of the 5′-phosphate.

[0162] Phosphate protecting groups include those described in U.S. Pat.Nos. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S.Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177,U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which isexpressly incorporated herein by reference in its entirety.

[0163] The oligonucleotides in accordance with this invention (singlestranded or double stranded) preferably comprise from about 8 to about80 nucleotides, more preferably from about 12-50 nucleotides and mostpreferably from about 15 to 30 nucleotides. As is known in the art, anucleotide is a base-sugar combination suitably bound to an adjacentnucleotide through a phosphodiester, phosphorothioate or other covalentlinkage.

[0164] The oligonucleotides of the present invention also includevariants in which a different base is present at one or more of thenucleotide positions in the oligonucleotide. For example, if the firstnucleotide is an adenosine, variants may be produced which containthymidine, guanosine or cytidine at this position. This may be done atany of the positions of the oligonucleotide. Thus, a 20-mer may comprise60 variations (20 positions×3 alternates at each position) in which theoriginal nucleotide is substituted with any of the three alternatenucleotides. These oligonucleotides are then tested using the methodsdescribed herein to determine their ability to inhibit expression ofp38α MAP kinase mRNA.

[0165] The oligonucleotides used in accordance with this invention maybe conveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives,including 2′-O-methoxyethyl oligonucleotides [Martin, P., Helv. Chim.Acta, 78, 486 (1995)]. It is also well known to use similar techniquesand commercially available modified amidites and controlled-pore glass(CPG) products such as biotin, fluorescein, acridine orpsoralen-modified amidites and/or CPG (available from Glen Research,Sterling Va.) to synthesize fluorescently labeled, biotinylated or otherconjugated oligonucleotides.

[0166] The antisense compounds of the present invention includebioequivalent compounds, including pharmaceutically acceptable salts andprodrugs. This is intended to encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids.

[0167] Pharmaceutically acceptable “salts” are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto [see,for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci.,66:1 (1977)].

[0168] For oligonucleotides, examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0169] The oligonucleotides of the invention may additionally oralternatively be prepared to be delivered in a “prodrug” form. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

[0170] For therapeutic or prophylactic treatment, oligonucleotides areadministered in accordance with this invention. Oligonucleotidecompounds of the invention may be formulated in a pharmaceuticalcomposition, which may include pharmaceutically acceptable carriers,thickeners, diluents, buffers, preservatives, surface active agents,neutral or cationic lipids, lipid complexes, liposomes, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients and the like in addition to the oligonucleotide.Such compositions and formulations are comprehended by the presentinvention.

[0171] Pharmaceutical compositions comprising the oligonucleotides ofthe present invention may include penetration enhancers in order toenhance the alimentary delivery of the oligonucleotides. Penetrationenhancers may be classified as belonging to one of five broadcategories, i.e., fatty acids, bile salts, chelating agents, surfactantsand non-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, 8:91-192; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7:1). One or more penetrationenhancers from one or more of these broad categories may be included.

[0172] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional compatiblepharmaceutically-active materials such as, e.g., antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the composition of present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the invention.

[0173] Regardless of the method by which the oligonucleotides of theinvention are introduced into a patient, colloidal dispersion systemsmay be used as delivery vehicles to enhance the in vivo stability of theoligonucleotides and/or to target the oligonucleotides to a particularorgan, tissue or cell type. Colloidal dispersion systems include, butare not limited to, macromolecule complexes, nanocapsules, microspheres,beads and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, liposomes and lipid:oligonucleotide complexesof uncharacterized structure. A preferred colloidal dispersion system isa plurality of liposomes. Liposomes are microscopic spheres having anaqueous core surrounded by one or more outer layers made up of lipidsarranged in a bilayer configuration [see, generally, Chonn et al.,Current Op. Biotech., 6, 698 (1995)].

[0174] The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer, metered dose inhaler or dry powder inhaler; intratracheal,intranasal, or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0175] Formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

[0176] Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

[0177] Compositions for parenteral administration may include sterileaqueous solutions which may also contain buffers, diluents and othersuitable additives. In some cases it may be more effective to treat apatient with an oligonucleotide of the invention in conjunction withother traditional therapeutic modalities in order to increase theefficacy of a treatment regimen. In the context of the invention, theterm “treatment regimen” is meant to encompass therapeutic, palliativeand prophylactic modalities. For example, a patient may be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,taxol,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., eds.,Rahay, N.J., 1987). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

[0178] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0179] Thus, in the context of this invention, by “therapeuticallyeffective amount” is meant the amount of the compound which is requiredto have a therapeutic effect on the treated mammal. This amount, whichwill be apparent to the skilled artisan, will depend upon the type ofmammal, the age and weight of the mammal, the type of disease to betreated, perhaps even the gender of the mammal, and other factors whichare routinely taken into consideration when treating a mammal with adisease. A therapeutic effect is assessed in the mammal by measuring theeffect of the compound on the disease state in the animal. For example,if the disease to be treated is cancer, therapeutic effects are assessedby measuring the rate of growth or the size of the tumor, or bymeasuring the production of compounds such as cytokines, production ofwhich is an indication of the progress or regression of the tumor.

[0180] The following examples illustrate the present invention and arenot intended to limit the same.

EXAMPLES

[0181] Eample 1

Synthesis of Oligonucleotides

[0182] Unmodified oligodeoxynucleotides are synthesized on an automatedDNA synthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

[0183] 2′-methoxy oligonucleotides are synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2′-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va.

[0184] 2′-fluoro oligonucleotides are synthesized as described inKawasaki et al., J. Med. Chem., 36, 831 (1993). Briefly, the protectednucleoside N⁶-benzoyl-2′-deoxy-2′-fluoroadenosine is synthesizedutilizing commercially available 9-β-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-α-fluoro atom is introduced by a S_(N)2-displacement of a2′-β-O-trifyl group. Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine isselectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl(THP) intermediate. Deprotection of the THP and N⁶-benzoyl groups isaccomplished using standard methodologies and standard methods are usedto obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramiditeintermediates.

[0185] The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-β-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group is followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation is followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies are used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0186] Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by themodification of a known procedure in which2,2′-anhydro-1-B-D-arabinofuranosyluracil is treated with 70% hydrogenfluoride-pyridine. Standard procedures are used to obtain the 5′-DMT and5′-DMT-3′phosphoramidites.

[0187] 2′-deoxy-2′-fluorocytidine is synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures are used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0188] 2′-(2-methoxyethyl)-modified amidites were synthesized accordingto Martin, P., Helv. Chim. Acta, 78,486 (1995). For ease of synthesis,the last nucleotide was a deoxynucleotide. 2′-O—CH₂CH₂OCH₃-cytosines maybe 5-methyl cytosines. Synthesis of 5-Methyl Cytosine Monomers:

[0189] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

[0190] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 hours) to give a solid which was crushed to a light tanpowder (57 g, 85% crude yield). The material was used as is for furtherreactions.

[0191] 2′-O-Methoxyethyl-5-methyluridine:

[0192] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product.

[0193] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

[0194] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/Hexane/Acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0195]3′-O-Acetyl-2′-O-methoxyethyl-51-O-dimethoxytrityl-5-methyluridine:

[0196] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by tic by first quenching the tic sample with the additionof MeOH. Upon completion of the reaction, as judged by tic, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

[0197]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

[0198] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10EC, and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

[0199] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0200] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0201]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

[0202] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, tlc showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/-Hexane (1:1) containing 0.5% Et₃NHas the eluting solvent. The pure product fractions were evaporated togive 90 g (90%) of the title compound.

[0203]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

[0204]N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (tlc showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0205] 5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotideswere synthesized according to published methods [Sanghvi et al., Nucl.Acids Res., 21, 3197 (1993)] using commercially availablephosphoramidites (Glen Research, Sterling Va. or ChemGenes, NeedhamMass.).

[0206] 2=-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0207] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0208] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0209] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013eq, 0.0054mmol) are dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1eq, 0.458 mmol) isadded in one portion. The reaction is stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicates a complete reaction.The solution is concentrated under reduced pressure to a thick oil. Thisis partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer is dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil is dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution is cooled to −10° C. The resulting crystallineproduct is collected by filtration, washed with ethyl ether (3×200 mL)and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLCand NMR are used to check consistency with pure product.

[0210]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0211] In a 2 L stainless steel, unstirred pressure reactor is addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) is addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) are added with manualstirring. The reactor is sealed and heated in an oil bath until aninternal temperature of 160° C. is reached and then maintained for 16 h(pressure<100 psig). The reaction vessel is cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicates % conversion to the product. In orderto avoid additional side product formation, the reaction is stopped,concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath(40-100° C.) with the more extreme conditions used to remove theethylene glycol. [Alternatively, once the low boiling solvent is gone,the remaining solution can be partitioned between ethyl acetate andwater. The product will be in the organic phase.] The residue ispurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions arecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. TLC and NMR are used to determine consistency with pureproduct.

[0212]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0213]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819, 86%).

[0214]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0215]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) is dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) is added dropwise at −10° C. to 0°C. After 1 hr the mixture is filtered, the filtrate is washed with icecold CH₂Cl₂ and the combined organic phase is washed with water, brineand dried over anhydrous Na₂SO₄. The solution is concentrated to get2′-O-(aminooxyethyl) thymidine, which is then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1eg.) is addedand the mixture for 1 hr. Solvent is removed under vacuum; residuechromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam.

[0216]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0217]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) is dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) is added to this solution at 10° C. under inertatmosphere. The reaction mixture is stirred for 10 minutes at 10° C.After that the reaction vessel is removed from the ice bath and stirredat room temperature for 2 hr, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) is added and extracted withethyl acetate (2×20 mL). Ethyl acetate phase is dried over anhydrousNa₂SO₄, evaporated to dryness. Residue is dissolved in a solution of 1MPPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) isadded and the reaction mixture is stirred at room temperature for 10minutes. Reaction mixture cooled to 10° C. in an ice bath, sodiumcyanoborohydride (0.39 g, 6.13 mmol) is added and reaction mixturestirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixtureis removed from the ice bath and stirred at room temperature for 2 hrs.To the reaction mixture 5% NaHCO₃ (25 mL) solution is added andextracted with ethyl acetate (2×25 mL). Ethyl acetate layer is driedover anhydrous Na₂SO₄ and evaporated to dryness. The residue obtained ispurified by flash column chromatography and eluted with 5% MeOH inCH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g).

[0218] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0219] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) is dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF is then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionis monitored by TLC (5% MeOH in CH₂Cl₂). Solvent is removed under vacuumand the residue placed on a flash column and eluted with 10% MeOH inCH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg).

[0220] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0221] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)is dried over P₂O₅ under high vacuum overnight at 40° C. It is thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained isdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) is added to the mixture and the reactionmixture is stirred at room temperature until all of the startingmaterial disappeared. Pyridine is removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.13 g).

[0222]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0223] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) is co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) is added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture is dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹′-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) is added. The reaction mixture is stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent is evaporated,then the residue is dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer is dried over anhydrousNa₂SO₄ and concentrated. Residue obtained is chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g).

[0224] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0225] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0226]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0227] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0228] Oligonucleotides having methylene (methylimino) (MMI) backbonesare synthesized according to U.S. Pat. No. 5,378,825, which iscoassigned to the assignee of the present invention and is incorporatedherein in its entirety. For ease of synthesis, various nucleoside dimerscontaining MMI linkages were synthesized and incorporated intooligonucleotides. Other nitrogen-containing backbones are synthesizedaccording to WO 92/20823 which is also coassigned to the assignee of thepresent invention and incorporated herein in its entirety.

[0229] Oligonucleotides having amide backbones are synthesized accordingto De Mesmaeker et al., Acc. Chem. Res., 28, 366 (1995). The amidemoiety is readily accessible by simple and well-known synthetic methodsand is compatible with the conditions required for solid phase synthesisof oligonucleotides.

[0230] Oligonucleotides with morpholino backbones are synthesizedaccording to U.S. Pat. No. 5,034,506 (Summerton and Weller).

[0231] Peptide-nucleic acid (PNA) oligomers are synthesized according toP. E. Nielsen et al., Science, 254, 1497 (1991).

[0232] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹P nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al., J. Biol. Chem., 266, 18162 (1991).Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

Example 2 Human p38α Oligonucleotide Sequences

[0233] Antisense oligonucleotides were designed to target human p38α.Target sequence data are from the p38 MAPK cDNA sequence; Genbankaccession number L35253, provided herein as SEQ ID NO: 1.Oligonucleotides was synthesized as chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of eight 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by six-nucleotide “wings.” The wings are composedof 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. All2′-MOE cytosines were 5-methylcytosines. These oligonucleotide sequencesare shown in Table 1.

[0234] The human Jurkat T-cell line (American Type Culture Collection,Manassas, Va.) was maintained in RPMI 1640 growth media supplementedwith 10% fetal bovine serum (FBS; Hyclone, Logan, Utah). HUVEC cells(Clonetics, San Diego, Calif.) were cultivated in endothelial basalmedia supplemented with 10% FBS (Hyclone, Logan, Utah).

[0235] Jurkat cells were grown to approximately 75% confluency andresuspended in culture media at a density of 1×10⁷ cells/ml. A total of3.6×10⁶ cells were employed for each treatment by combining 360 μl ofcell suspension with oligonucleotide at the indicated concentrations toreach a final volume of 400 μl. Cells were then transferred to anelectroporation cuvette and electroporated using an ElectrocellManipulator 600 instrument (Biotechnologies and Experimental Research,Inc.) employing 150 V, 1000 μF, at 13 Ω. Electroporated cells were thentransferred to conical tubes containing 5 ml of culture media, mixed byinversion, and plated onto 10 cm culture dishes.

[0236] HUVEC cells were allowed to reach 75% confluency prior to use.The cells were washed twice with warm (37° C.) OPTI-MEM™ (LifeTechnologies). The cells were incubated in the presence of theappropriate culture medium, without the growth factors added, and theoligonucleotide formulated in LIPOFECTIN7 (Life Technologies), a 1:1(w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water.HUVEC cells were treated with 100 nM oligonucleotide in 10 μg/mlLIPOFECTIN7. Treatment was for four hours.

[0237] Total mRNA was isolated using the RNEASY7 Mini Kit (Qiagen,Valencia, Calif.; similar kits from other manufacturers may also beused), separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane(Amersham Pharmacia Biotech, Piscataway, N.J.), a positively chargednylon membrane, and probed. p38 MAPK probes were made using thePrime-A-Gene7 kit (Promega Corporation, Madison, Wis.), a random primerlabeling kit, using mouse p38α or p38β cDNA as a template. Aglyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was purchasedfrom Clontech (Palo Alto, Calif.), Catalog Number 9805-1. The fragmentswere purified from low-melting temperature agarose, as described inManiatis, T., et al., Molecular Cloning: A Laboratory Manual, 1989. TheG3PDH probe was labeled with REDIVUE™ ³²P-dCTP (Amersham PharmaciaBiotech, Piscataway, N.J.) and Strip-EZ labelling kit (Ambion, Austin,Tex.). mRNA was quantitated by a PhosphoImager (Molecular Dynamics,Sunnyvale, Calif.). TABLE 1 Nucleotide Sequences of Human p38α Chimeric(deoxy gapped) Phosphorothioate Oligonucleotides SEQ TARGET GENE GENEISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ → 3′) NO:CO-ORDINATES² REGION 16486 AAGACCGGGCCCGGAATTCC 3 0001-0020 5′-UTR 16487GTGGAGGCCAGTCCCCGGGA 4 0044-0063 5′-UTR 16488 TGGCAGCAAAGTGCTGCTGG 50087-0106 5′-UTR 16489 CAGAGAGCCTCCTGGGAGGG 6 0136-0155 5′-UTR 16490TGTGCCGAATCTCGGCCTCT 7 0160-0179 5′-UTR 16491 GGTCTCGGGCGACCTCTCCT 80201-0220 5′-UTR 16492 CAGCCGCGGGACCAGCGGCG 9 0250-0269 5′-UTR 16493CATTTTCCAGCGGCAGCCGC 10 0278-0297 AUG 16494 TCCTGAGACATTTTCCAGCG 110286-0305 AUG 16495 CTGCCGGTAGAACGTGGGCC 12 0308-0327 coding 16496GTAAGCTTCTGACATTTCAC 13 0643-0662 coding 16497 TTTAGGTCCCTGTGAATTAT 140798-0817 coding 16498 ATGTTCTTCCAGTCAACAGC 15 0939-0958 coding 16499TAAGGAGGTCCCTGCTTTCA 16 1189-1208 coding 16500 AACCAGGTGCTCAGGACTCC 171368-1387 stop 16501 GAAGTGGGATCAACAGAACA 18 1390-1409 3′-UTR 16502TGAAAAGGCCTTCCCCTCAC 19 1413-1432 3′-UTR 16503 AGGCACTTGAATAATATTTG 201444-1463 3′-UTR 16504 CTTCCACCATGGAGGAAATC 21 1475-1494 3′-UTR 16505ACACATGCACACACACTAAC 22 1520-1539 3′-UTR

[0238] For an initial screen of human p38α antisense oligonucleotides,Jurkat cells were electroporated with 10 μM oligonucleotide. mRNA wasmeasured by Northern blot. Results are shown in Table 2.Oligonucleotides 16496 (SEQ ID NO. 13), 16500 (SEQ ID NO. 17) and 16503(SEQ ID NO. 20) gave 35% or greater inhibition of p38α mRNA. TABLE 2Inhibition of Human p38α mRNA expression in Jurkat Cells by Chimeric(deoxy gapped) Phosphorothioate Oligonucleotides SEQ GENE ISIS ID TARGET% mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION control — — 100%  0%16486  3 5′-UTR 212% — 16487  4 5′-UTR 171% — 16488  5 5′-UTR 157% —16489  6 5′-UTR 149% — 16490  7 5′-UTR 152% — 16491  8 5′-UTR 148% —16492  9 5′-UTR 125% — 16493 10 AUG 101% — 16494 11 AUG  72% 28% 1649512 coding  72% 28% 16496 13 coding  61% 39% 16497 14 coding 104% — 1649815 coding  88% 12% 16499 16 coding  74% 26% 16500 17 stop  63% 37% 1650118 3′-UTR  77% 23% 16502 19 3′-UTR  79% 21% 16503 20 3′-UTR  65% 35%16504 21 3′-UTR  72% 28% 16505 22 3′-UTR  93%  7%

[0239] The most active human p38α oligonucleotides were chosen for doseresponse studies. Oligonucleotide 16490 (SEQ ID NO. 7) which showed noinhibition in the initial screen was included as a negative control.Jurkat cells were grown and treated as described above except theconcentration of oligonucleotide was varied as indicated in Table 3.Results are shown in Table 3. Each of the active oligonucleotides showeda dose response effect with IC₅₀s around 10 nM. Maximum inhibition wasapproximately 70% with 16500 (SEQ ID NO 17). The most activeoligonucleotides were also tested for their ability to inhibit p38β.None of these oligonucleotides significantly reduced p38β mRNAexpression. TABLE 3 Dose Response of p38α mRNA in Jurkat cells to humanp38α Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ IDASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibitioncontrol — — — 100%   0% 16496 13 coding 2.5 nM 94%  6% ″ ″ ″   5 nM 74%26% ″ ″ ″  10 nM 47% 53% ″ ″ ″  20 nM 41% 59% 16500 17 stop 2.5 nM 82%18% ″ ″ ″   5 nM 71% 29% ″ ″ ″  10 nM 49% 51% ″ ″ ″  20 nM 31% 69% 1650320 3′-UTR 2.5 nM 74% 26% ″ ″ ″   5 nM 61% 39% ″ ″ ″  10 nM 53% 47% ″ ″ ″ 20 nM 41% 59% 16490  7 5′-UTR 2.5 nM 112%  — ″ ″ ″   5 nM 109%  — ″ ″ ″ 10 nM 104%  — ″ ″ ″  20 nM 97%  3%

Example 3 Human p38β Oligonucleotide Sequences

[0240] Antisense oligonucleotides were designed to target human p38β.Target sequence data are from the p38β MAPK cDNA sequence; Genbankaccession number U53442, provided herein as SEQ ID NO: 23.Oligonucleotides was synthesized as chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) throughout theoligonucleotide. All 2′-MOE cytosines were 5-methyl-cytosines. Theseoligonucleotide sequences are shown in Table 4. TABLE 4 NucleotideSequences of Human p38β Phosphorothioate Oligonucleotides SEQ TARGETGENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ → 3′)NO: CO-ORDINATES² REGION 17891 CGACATGTCCGGAGCAGAAT 25 0006-0025 AUG17892 TTCAGCTCCTGCCGGTAGAA 26 0041-0060 coding 17893TGCGGCACCTCCCACACGGT 27 0065-0084 coding 17894 CCGAACAGACGGAGCCGTAT 280121-0140 coding 17895 GTGCTTCAGGTGCTTGAGCA 29 0240-0259 coding 17896GCGTGAAGACGTCCAGAAGC 30 0274-0293 coding 17897 ACTTGACGATGTTGTTCAGG 310355-0374 coding 17898 AACGTGCTCGTCAAGTGCCA 32 0405-0424 coding 17899ATCCTGAGCTCACAGTCCTC 33 0521-0540 coding 17900 ACTGTTTGGTTGTAATGCAT 340635-0654 coding 17901 ATGATGCGCTTCAGCTGGTC 35 0731-0750 coding 17902GCCAGTGCCTCAGGTGCACT 36 0935-0954 coding 17903 AACGCTCTCATCATATGGCT 371005-1024 coding 17904 CAGCACCTCACTGCTCAATC 38 1126-1145 stop 17905TCTGTGACCATAGGAGTGTG 39 1228-1247 3′-UTR 17906 ACACATGTTTGTGCATGCAT 401294-1313 3′-UTR 17907 CCTACACATGGCAAGCACAT 41 1318-1337 3′-UTR 17908TCCAGGCTGAGCAGCTCTAA 42 1581-1600 3′-UTR 17909 AGTGCACGCTCATCCACACG 431753-1772 3′-UTR 17910 CTTGCCAGATATGGCTGCTG 44 1836-1855 3′-UTR

[0241] For an initial screen of human p38β antisense oligonucleotides,HUVEC cells were cultured and treated as described in Example 2. mRNAwas measured by Northern blot as described in Example 2. Results areshown in Table 5. Every oligonucleotide tested gave at least 50%inhibition. Oligonucleotides 17892 (SEQ ID NO. 26), 17893 (SEQ ID NO.27), 17894 (SEQ ID NO. 28), 17899 (SEQ ID NO. 33), 17901 (SEQ ID NO.35), 17903 (SEQ ID NO. 37), 17904 (SEQ ID NO. 38), 17905 (SEQ ID NO.39), 17907 (SEQ ID NO. 41), 17908 (SEQ ID NO. 42), and 17909 (SEQ ID NO.43) gave greater than approximately 85% inhibition and are preferred.TABLE 5 Inhibition of Human p38β mRNA expression in Huvec Cells byChimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ GENE ISISID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION control — —100%   0% 17891 25 AUG 22% 78% 17892 26 coding 10% 90% 17893 27 coding 4% 96% 17894 28 coding 13% 87% 17895 29 coding 25% 75% 17896 30 coding24% 76% 17897 31 coding 25% 75% 17898 32 coding 49% 51% 17899 33 coding 5% 95% 17900 34 coding 40% 60% 17901 35 coding 15% 85% 17902 36 coding49% 51% 17903 37 coding 11% 89% 17904 38 stop  9% 91% 17905 39 3′-UTR14% 86% 17906 40 3′-UTR 22% 78% 17907 41 3′-UTR  8% 92% 17908 42 3′-UTR17% 83% 17909 43 3′-UTR 13% 87% 17910 44 3′-UTR 26% 74%

[0242] Oligonucleotides 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO:33),17904 (SEQ ID NO. 38), and 17907 (SEQ ID NO. 41) were chosen for doseresponse studies. HUVEC cells were cultured and treated as described inExample 2 except that the oligonucleotide concentration was varied asshown in Table 6. The Lipofectin7/Oligo ratio was maintained at 3 μgLipofectin7/100 nM oligo, per ml. mRNA was measured by Northern blot asdescribed in Example 2.

[0243] Results are shown in Table 6. Each oligonucleotide tested had anIC₅₀ of less than 10 nM. The effect of these oligonucleotides on humanp38α was also determined. Only oligonucleotide 17893 (SEQ ID NO. 27)showed an effect on p38α mRNA expression. The IC₅₀ of thisoligonucleotide was approximately 4 fold higher for p38α compared top38β. TABLE 6 Dose Response of p38β in Huvec cells to human p38βChimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ ID ASOGene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control— — — 100%   0% 17893 27 coding  10 nM 37% 63% ″ ″ ″  25 nM 18% 82% ″ ″″  50 nM 16% 84% ″ ″ ″ 100 nM 19% 81% 17899 33 coding  10 nM 37% 63% ″ ″″  25 nM 23% 77% ″ ″ ″  50 nM 18% 82% ″ ″ ″ 100 nM 21% 79% 17904 38 stop 10 nM 31% 69% ″ ″ ″  25 nM 21% 79% ″ ″ ″  50 nM 17% 83% ″ ″ ″ 100 nM19% 81% 17907 41 3′-UTR  10 nM 37% 63% ″ ″ ″  25 nM 22% 78% ″ ″ ″  50 nM18% 72% ″ ″ ″ 100 nM 18% 72%

Example 4 Rat p38α Oligonucleotide Sequences

[0244] Antisense oligonucleotides were designed to target rat p38α.Target sequence data are from the p38 MAPK cDNA sequence; Genbankaccession number U73142, provided herein as SEQ ID NO: 45.Oligonucleotides was synthesized as chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages in the wings are phosphodiester (P═O).Internucleoside linkages in the central gap are phosphorothioate (P═S).All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. Theseoligonucleotide sequences are shown in Table 7.

[0245] bEND.3, a mouse endothelial cell line (gift of Dr. Werner Risau;see Montesano et al., Cell, 1990, 62, 435, and Stepkowski et al., J.Immunol., 1994, 153, 5336) were grown in high-glucose DMEM (LifeTechnologies, Gaithersburg, Md.) medium containing 10% fetal bovineserum (FBS) and 1% Penicillin/Streptomycinin. Cells were plated atapproximately 2×10⁵ cells per 100 mm dish. Within 48 hours of plating,the cells were washed with phosphate-buffered saline (LifeTechnologies). Then, Opti-MEM7 medium containing 3 μg/mL LIPOFECTIN⁷ andan appropriate amount of oligonucleotide were added to the cells. As acontrol, cells were treated with LIPOFECTIN⁷ without oligonucleotideunder the same conditions and for the same times as theoligonucleotide-treated samples.

[0246] After 4 hours at 37° C., the medium was replaced with highglucose DMEM medium containing 10% FBS and 1% Penicillin/Streptomycinin.The cells were typically allowed to recover overnight (about 18 to 24hours) before RNA and/or protein assays were performed as described inExample 2. The p38α, p38β and G3PDH probes used were identical to thosedescribed in Example 2. TABLE 7 Nucleotide Sequences of Rat p38αPhosphorothioate Oligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDESEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ → 3′) NO CO-ORDINATES² REGION21844 CoToGoCoGsAsCsAsTsTsTsTsCsCsAsGoCoGoGoC 47 0001-0020 AUG 21845GoGoToAoAsGsCsTsTsCsTsGsAsCsAsCoToToCoA 48 0361-0380 coding 21846GoGoCoCoAsGsAsGsAsCsTsGsAsAsTsGoToAoGoT 49 0781-0800 coding 21871CoAoToCoAsTsCsAsGsGsGsTsCsGsTsGoGoToAoC 50 0941-0960 coding 21872GoGoCoAoCsAsAsAsGsCsTsAsAsTsGsAoCoToToC 51 1041-1060 coding 21873AoGoGoToGsCsTsCsAsGsGsAsCsTsCsCoAoToToT 52 1081-1100 stop 21874GoGoAoToGsGsAsCsAsGsAsAsCsAsGsAoAoGoCoA 53 1101-1120 3′-UTR 21875GoAoGoCoAsGsGsCsAsGsAsCsTsGsCsCoAoAoGoG 54 1321-1340 3′-UTR 21876AoGoGoCoTsAsGsAsGsCsCsCsAsGsGsAoGoCoCoA 55 1561-1580 3′-UTR 21877GoAoGoCoCsTsGsTsGsCsCsTsGsGsCsAoCoToGoG 56 1861-1880 3′-UTR 21878ToGoCoAoCsCsAsCsAsAsGsCsAsCsCsToGoGoAoG 57 2081-2100 3′-UTR 21879GoGoCoToAsCsCsAsTsGsAsGsTsGsAsGoAoAoGoA 58 2221-2240 3′-UTR 21880GoToCoCoCsTsGsCsAsCsTsGsAsTsAsGoAoGoAoA 59 2701-2720 3′-UTR 21881ToCoToToCsCsAsAsTsGsGsAsGsAsAsAoCoToGoG 60 3001-3020 3′-UTR

[0247]¹Emboldened residues, 2′-methoxyethoxy-residues (others are2′-deoxy-); 2′-MOE cytosines and 2′-deoxy cytosine residues are5-methyl-cytosines; “s” linkages are phosphorothioate linkages; “o”linkages are phosphodiester linkages. ² Co-ordinates from GenbankAccession No. U73142, locus name “RNU73142”, SEQ ID NO. 45.

[0248] Rat p38α antisense oligonucleotides were screened in bEND.3 cellsfor inhibition of p38α and p38β mRNA expression. The concentration ofoligonucleotide used was 100 nM. Results are shown in Table 8.Oligonucleotides 21844 (SEQ ID NO. 47), 21845 (SEQ ID NO. 48), 21872(SEQ ID NO. 51), 21873 (SEQ ID NO. 52), 21875 (SEQ ID NO. 54), and 21876(SEQ ID NO.

[0249] 55) showed greater than approximately 70% inhibition of p38α mRNAwith minimal effects on p38β mRNA levels. Oligonucleotide 21871 (SEQ IDNO. 50) inhibited both p38α and p38β levels greater than 70%. TABLE 8Inhibition of Mouse p38 mRNA expression in bEND.3 Cells by Chimeric(deoxy gapped) Mixed Backbone p38α Antisense Oligonucleotides SEQ GENEISIS ID TARGET % p38α mRNA % p38β mRNA No: NO: REGION INHIBITIONINHIBITION control — —  0%  0% 21844 47 AUG 81% 20% 21845 48 coding 75%25% 21871 50 coding 90% 71% 21872 51 coding 87% 23% 21873 52 stop 90% 3% 21874 53 3′-UTR 38% 21% 21875 54 3′-UTR 77% — 21876 55 3′-UTR 69% —21877 56 3′-UTR 55% 13% 21878 57 3′-UTR 25% 10% 21879 58 3′-UTR — —21881 60 3′-UTR — —

[0250] Several of the most active oligonucleotides were selected fordose response studies. bEND.3 cells were cultured and treated asdescribed above, except that the concentration of oligonucleotide wasvaried as noted in Table 9. Results are shown in Table 9. TABLE 9 DoseResponse of bEND.3 cells to rat p38β Chimeric (deoxy gapped)Phosphorothioate Oligonucleotides SEQ ID ASO Gene % p38α mRNA % p38βmRNA ISIS # NO: Target Dose Inhibition Inhibition control — — — 100%  0% 21844 47 AUG  1 nM — — ″ ″ ″  5 nM — — ″ ″ ″  25 nM 36%  8% ″ ″ ″100 nM 80%  5% 21871 50 coding  1 nM  1% — ″ ″ ″  5 nM 23%  4% ″ ″ ″  25nM 34% 24% ″ ″ ″ 100 nM 89% 56% 21872 51 stop  1 nM — — ″ ″ ″  5 nM — —″ ″ ″  25 nM 35% — ″ ″ ″ 100 nM 76%  1% 21873 52 stop  1 nM — 53% ″ ″ ″ 5 nM — 31% ″ ″ ″  25 nM 54% 28% ″ ″ ″ 100 nM 92% 25% 21875 54 3′-UTR  1nM — 11% ″ ″ ″  5 nM — 16% ″ ″ ″  25 nM 33%  2% ″ ″ ″ 100 nM 72%  4%

Example 5 Mouse p38β Oligonucleotide Sequences

[0251] Antisense oligonucleotides were designed to target mouse p38β.Target sequence data are from a mouse EST sequence; Genbank accessionnumber AI119044, provided herein as SEQ ID NO 61. Oligonucleotides wassynthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides inlength, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings.” The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages in the wings are phosphodiester (P═O). Internucleoside linkagesin the central gap are phosphorothioate (P═S). All 2′-MOE cytosines and2′-OH cytosines were 5-methyl-cytosines. These oligonucleotide sequencesare shown in Table 10. TABLE 10 Nucleotide Sequences of Mouse p38βChimeric (deoxy gapped) Phosphorothioate Oligonucleotides TARGET SEQGENE ISIS NUCLEOTIDE SEQUENCE′ ID NUCLEOTIDE NO. (5′ → 3′) NO:CO-ORDINATES² 100800 CoAoCoAoGsAsAsGsCsAsGsCsTsGsGsAoGoCoGoA 630051-0070 100801 ToGoCoGoGsCsAsCsCsTsCsCsCsAsTsAoCoToGoT 64 0119-0138100802 CoCoCoToGsCsAsGsCsCsGsCsTsGsCsGoGoCoAoC 65 0131-0150 100803GoCoAoGoAsCsTsGsAsGsCsCsGsTsAsGoGoCoGoC 66 0171-0190 100804ToToAoCoAsGsCsCsAsCsCsTsTsCsTsGoGoCoGoC 67 0211-0230 100805GoToAoToGsTsCsCsTsCsCsTsCsGsCsGoToGoGoA 68 0261-0280 100806AoToGoGoAsTsGsTsGsGsCsCsGsGsCsGoToGoAoA 69 0341-0360 100807GoAoAoToTsGsAsAsCsAsTsGsCsTsCsAoToCoGoC 70 0441-0460 100808AoCoAoToTsGsCsTsGsGsGsCsTsTsCsAoGoGoToC 71 0521-0540 100809AoToCoCoTsCsAsGsCsTsCsGsCsAsGsToCoCoToC 72 0551-0570 100810ToAoCoCoAsCsCsGsTsGsTsGsGsCsCsAoCoAoToA 73 0617-0636 100811CoAoGoToTsTsAsGsCsAsTsGsAsTsCsToCoToGoG 74 0644-0663 100812CoAoGoGoCsCsAsCsAsGsAsCsCsAsGsAoToGoToC 75 0686-0705 100813CoCoToToCsCsAsGsCsAsGsTsTsCsAsAoGoCoCoA 76 0711-0730 101123CoAoGoCoAsCsCsAsTsGsGsAsCsGsCsGoGoAoAoC 77 21871 mismatch

[0252] Mouse p38β antisense sequences were screened in bEND.3 cells asdescribed in Example 4. Results are shown in Table 11.

[0253] Oligonucleotides 100800 (SEQ ID NO. 63), 100801 (SEQ ID NO. 64),100803 (SEQ ID NO. 66), 100804 (SEQ ID NO. 67), 100805 (SEQ ID NO. 68),100807 (SEQ ID NO. 70), 100808 (SEQ ID NO. 71), 100809 (SEQ ID NO. 72),100810 (SEQ ID NO. 73), 100811 (SEQ ID NO.74), and 100813 (SEQ ID NO.76) resulted in at least 50% inhibition of p38β mRNA expression.Oligonucleotides 100801 (SEQ ID NO.64), 100803 (SEQ ID NO. 66), 100804(SEQ ID NO. 67), 100805 (SEQ ID NO. 68), 100809 (SEQ ID NO. 72), and100810 (SEQ ID NO. 73) resulted in at least 70% inhibition and arepreferred. Oligonucleotides 100801 (SEQ ID NO. 64), 100805 (SEQ ID NO.68), and 100811 (SEQ ID NO. 74) resulted in significant inhibition ofp38α mRNA expression in addition to their effects on p38β. TABLE 11Inhibition of Mouse p38 mRNA expression in bEND.3 Cells by Chimeric(deoxy gapped) Mixed Backbone p38β Antisense Oligonucleotides ISIS SEQID % p38β mRNA % p38α mRNA No: NO: INHIBITION INHIBITION control —  0% 0% 100800 63 51% — 100801 64 74% 31% 100802 65 35% — 100803 66 74% 18%100804 67 85% 18% 100805 68 78% 58% 100806 69 22%  3% 100807 70 64% —100808 71 53% 13% 100809 72 84% 14% 100810 73 72%  1% 100811 74 60% 43%100812 75 36% 17% 100813 76 54% —

Example 6 Effect of p38 MAPK Antisense Oligonucleotides on IL-6Secretion

[0254] p38 MAPK antisense oligonucleotides were tested for their abilityto reduce IL-6 secretion. bEND.3 cells were cultured and treated asdescribed in Example 4 except that 48 hours after oligonucleotidetreatment, cells were stimulated for 6 hours with 1 ng/mL recombinantmouse IL-1 (R&D Systems, Minneapolis, Minn.). IL-6 was measured in themedium using an IL-6 ELISA kit (Endogen Inc., Woburn, Mass.).

[0255] Results are shown in Table 12. Oligonucleotides targeting aspecific p38 MAPK isoform were effective in reducing IL-6 secretiongreater than approximately 50%. TABLE 12 Effect of p38 AntisenseOligonucleotides on IL-6 secretion ISIS SEQ ID DOSE % IL-6 No: NO: GENETARGET (μM) INHIBITION control — —  0%  21873 52 p38α 100 49% 100804 67p38β 100 57%  21871 50 p38α and p38β 200 23%

Example 7 Activity of p38α Antisense Oligonucleotides in RatCardiomyocytes

[0256] Rat p38α antisense oligonucleotides were screened in Rat A-10cells. A-10 cells (American Type Culture Collection, Manassas, Va.) weregrown in high-glucose DMEM (Life Technologies, Gaithersburg, Md.) mediumcontaining 10% fetal calf serum (FCS). Cells were treated witholigonucleotide as described in Example 2. Oligonucleotide concentrationwas 200 nM. mRNA was isolated 24 hours after time zero and quantitatedby Northern blot as described in Example 2.

[0257] Results are shown in Table 13. Oligonucleotides 21845 (SEQ ID NO.48), 21846 (SEQ ID NO. 49), 21871 (SEQ ID NO. 50), 21872 (SEQ ID NO.51), 21873 (SEQ ID NO. 52), 21874 (SEQ ID NO. 53), 21875 (SEQ ID NO.54), 21877 (SEQ ID NO. 56), 21878 (SEQ ID NO. 57), 21879 (SEQ ID NO.58), and 21881 (SEQ ID NO. 60) inhibited p38α mRNA expression by 65% orgreater in this assay. Oligonucleotides 21846 (SEQ ID NO. 49), 21871(SEQ ID NO. 50), 21872 (SEQ ID NO. 51), 21877 (SEQ ID NO. 56), and 21879(SEQ ID NO. 58) inhibited p38α mRNA expression by greater than 85% andare preferred. TABLE 13 Inhibition of Rat p38α mRNA expression in A-10Cells by Chimeric (deoxy gapped) Mixed Backbone p38α AntisenseOligonucleotides SEQ GENE ISIS ID TARGET % p38α mRNA % p38α mRNA No: NO:REGION EXPRESSION INHIBITION control — — 100%   0% 21844 47 AUG 75% 25%21845 48 coding 25% 75% 21846 49 coding  8% 92% 21871 50 coding 12% 88%21872 51 coding 13% 87% 21873 52 stop 19% 81% 21874 53 3′-UTR 22% 78%21875 54 3′-UTR 26% 74% 21876 55 3′-UTR 61% 39% 21877 56 3′-UTR 12% 88%21878 57 3′-UTR 35% 65% 21879 58 3′-UTR 11% 89% 21881 60 3′-UTR 31% 69%

[0258] The most active oligonucleotide in this screen (SEQ ID NO. 49)was used in rat cardiac myocytes prepared from neonatal rats (Zechner,D., et. al., J. Cell Biol., 1997, 139, 115-127). Cells were grown asdescribed in Zechner et al. and transfected with oligonucleotide asdescribed in Example 2. Oligonucleotide concentration was 1 μM. mRNA wasisolated 24 hrs after time zero and quantitated using Northern blottingas described in Example 2. An antisense oligonucleotide targeted toJNK-2 was used as a non-specific target control.

[0259] Results are shown in Table 14. Oligonucleotide 21846 (SEQ ID NO.49) was able to reduce p38α expression in rat cardiac myocytes by nearly60%. The JNK-2 antisense oligonucleotide had little effect on p38αexpression. TABLE 14 Inhibition of Rat p38α mRNA expression in RatCardiac Myocytes by A Chimeric (deoxy gapped) Mixed Backbone p38αAntisense Oligonucleotide SEQ GENE ISIS ID TARGET % p38α mRNA % p38αmRNA No: NO: REGION EXPRESSION INHIBITION control — — 100%  0% 21846 49coding  41% 59%

[0260] Eample 8

Additional Human p38α Oligonucleotide Sequences

[0261] Additional antisense oligonucleotides were designed to targethuman p38α based on active rat sequences. Target sequence data are fromthe p38 MAPK cDNA sequence; Genbank accession number L35253, providedherein as SEQ ID NO: 1. Oligonucleotides were synthesized as chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All 2′-MOE cytosines and 2′-OH cytosineswere 5-methyl-cytosines. These oligonucleotide sequences are shown inTable 15. TABLE 15 Additional Nucleotide Sequences of Human p38αChimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ TARGETGENE GENE ISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ → 3′)NO: CO-ORDINATES² REGION 100860 CTGAGACATTTTCCAGCGGC 78 0284-0303 Start100861 ACGCTCGGGCACCTCCCAGA 79 0344-0363 coding 100862AGCTTCTTCACTGCCACACG 80 0439-0458 coding 100863 AATGATGGACTGAAATGGTC 810464-0483 coding 100864 TCCAACAGACCAATCACATT 82 0538-0557 coding 100865TGTAAGCTTCTGACATTTCA 83 0644-0663 coding 100866 TGAATGTATATACTTTAGAC 840704-0723 coding 100867 CTCACAGTCTTCATTCACAG 85 0764-0783 coding 100868CACGTAGCCTGTCATTTCAT 86 0824-0843 coding 100869 CATCCCACTGACCAAATATC 870907-0926 coding 100870 TATGGTCTGTACCAGGAAAC 88 0960-0979 coding 100871AGTCAAAGACTGAATATAGT 89 1064-1083 coding 100872 TTCTCTTATCTGAGTCCAAT 901164-1183 coding 100873 CATCATCAGGATCGTGGTAC 91 1224-1243 coding 100874TCAAAGGACTGATCATAAGG 92 1258-1277 coding 100875 GGCACAAAGCTGATGACTTC 931324-1343 coding 100876 AGGTGCTCAGGACTCCATCT 94 1364-1383 stop 100877GCAACAAGAGGCACTTGAAT 95 1452-1471 3′-UTR

[0262] For an initial screen of human p38α antisense oligonucleotides,T-24 cells, a human transitional cell bladder carcinoma cell line, wereobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis. Acontrol oligonucleotide ISIS 118965 (TTATCCTAGCTTAGACCTAT, hereinincorporated as SEQ ID NO: 96) was synthesized as chimericoligonucleotide (“gapmer”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All 2′-MOE cytosines and 2′OH cytosineswere 5-methyl-cytosines.

[0263] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide. mRNA wasmeasured by Northern blot. Results are shown in Table 16.Oligonucleotides 100861 (SEQ ID NO. 79) 100862 (SEQ ID NO. 80), 100863(SEQ ID NO. 81), 100866 (SEQ ID NO. 84), 100867 (SEQ ID NO. 85), 100868(SEQ ID NO. 86) 100870 (SEQ ID NO. 88), 100871 (SEQ ID NO. 89), 100872(SEQ NO. 90), 100873 (SEQ ID NO. 91), and 100874 (SEQ ID NO. 92) 100875(SEQ ID NO. 93) and 100877 (SEQ ID NO. 95) gave greater thanapproximately 40% inhibition and are preferred. TABLE 16 Inhibition ofHuman p38α mRNA expression in T-24 Cells by Chimeric (deoxy gapped)Phosphorothioate Oligonucleotides SEQ ISIS ID GENE TARGET % P38α mRNA %P38β mRNA No: NO: REGION EXPRESSION EXPRESSION 100860 78 0284-0303 73%71% 100861 79 0344-0363 60% 47% 100862 80 0439-0458 56% 45% 100863 810464-0483 49% 67% 100864 82 0538-0557 66% 70% 100865 83 0644-0663 64%63% 100866 84 0704-0723 55% 65% 100867 85 0764-0783 58% 33% 100868 860824-0843 47% 60% 100869 87 0907-0926 61% 100%  100870 88 0960-0979 51%No data 100871 89 1064-1083 57% 96% 100872 90 1164-1183 37% 77% 10087391 1224-1243 34% 70% 100874 92 1258-1277 42% 76% 100875 93 1324-1343 39%90% 100876 94 1364-1383 77% 93% 100877 95 1452-1471 47% 95%

[0264] Oligonucleotides 100872 (SEQ ID NO. 90), 100873 (SEQ ID NO. 91),100874 (SEQ ID NO. 92), and 100875 (SEQ ID NO. 93) were chosen for doseresponse studies.

[0265] Results are shown in Table 17. The effect of theseoligonucleotides on human p38β was also determined. TABLE 17 DoseResponse of p38α in T-24 cells to human p38α Chimeric (deoxy gapped)Phosphorothioate Oligonucleotides SEQ ID ASO Gene % p38α mRNA % p38βmRNA ISIS # NO: Target Dose Expression Inhibition Control 96 — — 94% 80% 118965 100872 90 coding  50 nM 45% 108% ″ ″ ″ 100 nM 18% 91% ″ ″ ″200 nM 17% 92% 100873 91 coding  50 nM 19% 90% ″ ″ ″ 100 nM 12% 78% ″ ″″ 200 nM  8% 44% 100874 92 coding  50 nM 47% 107%  ″ ″ ″ 100 nM 27%101%  ″ ″ ″ 200 nM 13% 51% 100875 93 coding  50 nM 30% 105%  ″ ″ ″ 100nM 13% 92% ″ ″ ″ 200 nM  8% 69%

Example 9 Additional Human p38β Oligonucleotide Sequences

[0266] Additional antisense oligonucleotides were designed to targethuman p38β based on active rat sequences. Target sequence data are fromthe p38 MAPK cDNA sequence; Genbank accession number U53442, providedherein as SEQ ID NO: 23.

[0267] Oligonucleotides was synthesized as chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages in the wings are phosphodiester (P═O).Internucleoside linkages in the central gap are phosphorothioate (P═S).All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. Theseoligonucleotide sequences are shown in Table 18. A controloligonucleotide ISIS 118966 (GTTCGATCGGCTCGTGTCGA), herein incorporatedas SEQ ID NO: 107) was synthesized as chimeric oligonucleotide(“gapmer”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside(backbone) linkages are phosphorothioate (P═S) in the gap andphosphodiester in the wings. All 2′-MOE cytosines and 2′-OH cytosineswere 5-methyl-cytosines. TABLE 18 Additional Nucleotide Sequences ofHuman p38β Chimeric (deoxy gapped) Mixed-Backbone PhosphorothioateOligonucleotides SEQ TARGET GENE GENE ISIS NUCLEOTIDE SEQUENCE¹ IDNUCLEOTIDE TARGET NO. (5′ → 3′) NO: CO-ORDINATES² REGION 107869ACAGACGGAGCCGTAGGCGC 97 117-136 coding 107870 CACCGCCACCTTCTGGCGCA 98156-175 coding 107871 GTACGTTCTGCGCGCGTGGA 99 207-226 coding 107872ATGGACGTGGCCGGCGTGAA 100 287-306 coding 107873 CAGGAATTGAACGTGCTCGT 101414-433 coding 107874 ACGTTGCTGGGCTTCAGGTC 102 491-510 coding 107875TACCAGCGCGTGGCCACATA 103 587-606 coding 107876 CAGTTGAGCATGATCTCAGG 104614-633 coding 107877 CGGACCAGATATCCACTGTT 105 649-668 coding 107878TGCCCTGGAGCAGCTCAGCC 106 682-701 coding

[0268] For an initial screen of human p38β antisense oligonuleotides,T-24 cells, a human transitional cell bladder carcinoma cell line, wereobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis. Acontrol oligonucleotide ISIS 118966 (TTATCCTAGCTTAGACCTAT, hereinincorporated as SEQ ID NO: 106) was synthesized as chimericoligonucleotide (“gapmer”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.”The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S) in thegap and phosphodiester in the wings. All 2′-MOE cytosines and 2′-OHcytosines were 5-methyl-cytosines.

[0269] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide. mRNA wasmeasured by Northern blot. Results are shown in Table 19. Forcomparison, ISIS 17893 and ISIS 17899, both targeting human p38β (SEQ IDNO: 27) and ISIS 100802 targeting mouse p38β (SEQ ID NO: 65) describedin Examples 3 and 5 above, respectively, were included in the screen.

[0270] Oligonucleotides 107869 (SEQ ID NO. 97), 107871 (SEQ ID NO. 99),107872 (SEQ ID NO. 100), 107873 (SEQ ID NO. 101), 107878 (SEQ IDNO.106), 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO. 33) and 100802 (SEQ IDNO.65, targeted to mouse p38β) gave greater than approximately 40%inhibition and are preferred. TABLE 19 Inhibition of Human p38β mRNAexpression in T-24 Cells by Chimeric (deoxy gapped) Mixed-BackbonePhosphorothioate Oligonucleotides SEQ ISIS ID GENE TARGET % p38β mRNA %p38α mRNA No: NO: REGION EXPRESSION EXPRESSION 107869  97 Coding 60% 93% 107870  98 Coding 74%  97% 107871  99 Coding 60% 111% 107872 100Coding 57% 123% 107873 101 Coding 58% 120% 107874 102 Coding 61% 100%107875 103 Coding 92% 112% 107876 104 Coding 127%  137% 107877 105Coding No data No data 107878 106 Coding 54% 112%  17893  27 Coding 31% 61%  17899  33 Coding 56% 117% 100802  65 Coding 47%  78%

[0271] Oligonucleotides 107871, 107872, 107873, 107874, 107875, 107877,107878, 17893 and 17899 were chosen for dose response studies.

[0272] Results are shown in Table 20. The effect of theseoligonucleotides on human p38α was also determined. TABLE 20 DoseResponse of p38β in T-24 cells to human p38β Chimeric (deoxy gapped)Mixed-backbone Phosphorothioate Oligonucleotides SEQ ID ASO Gene % p38βmRNA % p38α mRNA ISIS # NO: Target Dose Expression Inhibition Control107 — — 100%  100% 118966 107871  99 coding  50 nM 41% 105% ″ ″ ″ 100 nM42% 132% ″ ″ ″ 200 nM 10% 123% 107872 100 coding  50 nM 71% 124% ″ ″ ″100 nM 13%  84% ″ ″ ″ 200 nM 22% 102% 107873 101 coding  50 nM 69% 132%″ ″ ″ 100 nM 41% 119% ″ ″ ″ 200 nM 23% 131% 107874 102 coding  50 nM 75%109% ″ ″ ″ 100 nM 34%  99% ″ ″ ″ 200 nM 23%  87% 107875 103 coding  50nM 82%  93% ″ ″ ″ 100 nM 38% 101% ″ ″ ″ 200 nM 40%  91% 107877 105coding  50 nM 50% 127% ″ ″ ″ 100 nM 34% 125% ″ ″ ″ 200 nM 22% 106%107878 106 coding  50 nM 70% 110% ″ ″ ″ 100 nM 43% 109% ″ ″ ″ 200 nM 27%116%  17893  27 coding  50 nM 28%  88% ″ ″ ″ 100 nM 27% 115% ″ ″ ″ 200nM 16% 108%  17899  33 coding  50 nM 89%  87% ″ ″ ″ 100 nM 36% 104% ″ ″″ 200 nM 15%  80%

[0273] These data show that the oligonucleotides designed to targethuman p38β, do so in a target-specific and dose-dependent manner.

Eample 10 Real-Time Quantitative PCR Analysis of p38β mRNA Levels

[0274] Quantitation of p38α mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

[0275] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0276] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution (20-200 ng). The RT reactionwas carried out by incubation for 30 minutes at 48° C. Following a 10minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles ofa two-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0277] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0278] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at485 nm and emission at 530 nm.

[0279] Probes and primers to human p38α were designed to hybridize to ahuman p38α sequence, using published sequence information (GenBankaccession number L35253, incorporated herein as SEQ ID NO:1). For humanp38α the PCR primers were:

[0280] forward primer: GATGAGTGGAAAAGCCTGAC (SEQ ID NO: 108)

[0281] reverse primer: CTGCAACAAGAGGCACTTGA (SEQ ID NO: 109) and the PCRprobe was: FAM-GATGAAGTCATCAGCTTTGTGCCACCACCCCTTGACCAAGAAGAGATGGA-TAMRA(SEQ ID NO: 110) where FAM is the fluorescent dye and TAMRA is thequencher dye. For human GAPDH the PCR primers were:

[0282] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 111)

[0283] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 112) and the PCRprobe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 113) whereJOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0284] Probes and primers to mouse p38α were designed to hybridize to amouse p38α sequence, using published sequence information (GenBankaccession number U10871.1, incorporated herein as SEQ ID NO: 114). Formouse p38α the PCR primers were:

[0285] forward primer: AAGGGAACGAGAAAACTGCTGTT (SEQ ID NO: 115)

[0286] reverse primer: TATTTTAACCAGTGGTATTATCTGACATCCT (SEQ ID NO: 116)and the PCR probe was: FAM-TTGTATTTGTGAACTTGGCTGTAATCTGGTATGCC -TAMRA

[0287] (SEQ ID NO: 117) where FAM is the fluorescent reporter dye andTAMRA is the quencher dye. For mouse GAPDH the PCR primers were:

[0288] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO: 118)

[0289] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 119) and the PCRprobe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 120)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0290] Probes and primers to rat p38α were designed to hybridize to arat p38α sequence, using published sequence information (GenBankaccession number U73142, incorporated herein as SEQ ID NO: 45). For ratp38α the PCR primers were:

[0291] forward primer: ATCATTTGGAGCCCAGAAGGA (SEQ ID NO: 121)

[0292] reverse primer: TGGAGCTGGACTGCATACTGA (SEQ ID NO: 122) and thePCR probe was: FAM-CTGGCCAGGCCTCACCGC-TAMRA

[0293] (SEQ ID NO: 123) where FAM is the fluorescent reporter dye andTAMRA is the quencher dye. For rat GAPDH the PCR primers were:

[0294] forward primer: TGTTCTAGAGACAGCCGCATCTT(SEQ ID NO: 124)

[0295] reverse primer: CACCGACCTTCACCATCTTGT(SEQ ID NO: 125) and the PCRprobe was: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 126)where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 11 Additional Human p38α Oligonucleotide Sequences

[0296] Additional antisense oligonucleotides were designed to targethuman p38α using published sequence (Genbank accession numberNM_(—)001315.1, provided herein as SEQ ID NO: 127). Oligonucleotideswere synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotidesin length, composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings. ” The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. Internucleoside linkages arephosphorothioate (P═S). These oligonucleotide sequences are shown inTable 21. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the compound binds. Thecompounds can be analyzed for their effect on human p38α mRNA levels byquantitative real-time PCR as described in other examples herein. TABLE21 Additional chimeric phosphorothioate antisense oligonucleotidestargeted to human p38α Target Sequence Target SEQ ISIS # RegionAccession # Site SEQUENCE ID NO: 186877 coding NM_001315.1 1271GAGCAAAGTAGGCATGTGCA 128 186878 3′ UTR NM_001315.1 2703GTTTCCGAAGTTTGGGATAT 129 186879 3′ UTR NM_001315.1 2735GCATTAGTTATTGGGAGTGA 130 186880 3′ UTR NM_001315.1 1671CCCTGGAGCATCCACAACCT 131 186881 coding NM_001315.1 1021TGTACCAGGAAACAATGTTC 132 186882 5′ UTR NM_001315.1 326CGGGCAAGAAGGTGGCCCTG 133 186883 3′ UTR NM_001315.1 3296ATCGCCATCAGTCTGCCTCC 134 186884 3′ UTR NM_001315.1 2312TGACATCAAGAACCTGCTTC 135 186885 3′ UTR NM_001315.1 2134GGCCCACAAGCAGCTGTCCA 136 186886 3′ UTR NM_001315.1 3063TGAAAACGACACTTCTCCAC 137 186887 3′ UTR NM_001315.1 3307GGTGAGAGGGAATCGCCATC 138 186888 3′ UTR NM_001315.1 2007ATACTGTCAAGATCTGAGAA 139 186889 3′ UTR NM_001315.1 2702TTTCCGAAGTTTGGGATATT 140 186890 3′ UTR NM_001315.1 2205AGAGAGACGCACATATACGC 141 186891 3′ UTR NM_001315.1 1516CAACAGGCACTTGAATAATA 142 186892 coding NM_001315.1 638ATTCCTCCAGAGACCTTGCA 143 186893 3′ UTR NM_001315.1 2848AAGACACCTTGTTACTTTTT 144 186894 3′ UTR NM_001315.1 2989TGCCCTTTCTCCCCATCAAA 145 186895 coding NM_001315.1 1096TGGCATCCTGTTAATGAGAT 146 186896 3′ UTR NM_001315.1 1477AAGGCCTTCCCCTCACAGTG 147 186897 3′ UTR NM_001315.1 3728AATAGGCTTTATTTTAACCA 148 186898 3′ UTR NM_001315.1 2455ACCCAAGAAGTCTTCACTGG 149 186899 3′ UTR NM_001315.1 3135TTTCTTATTACACAAAAGGC 150 186900 3′ UTR NM_001315.1 3445GGAAATCACACGAGCATTTA 151 186901 coding NM_001315.1 794GGTCCCTGTGAATTATGTCA 152 186902 3′ UTR NM_001315.1 3112AATATATGAGTCCTCATGTA 153 186903 3′ UTR NM_001315.1 3511CTAACACGTATGTGGTCACA 154 186904 3′ UTR NM_001315.1 2984TTTCTCCCCATCAAAAGGAA 155 186905 coding NM_001315.1 727CTGAACATGGTCATCTGTAA 156 186906 3′ UTR NM_001315.1 3681ATAACTGATTACAGCCAAGT 157 186907 3′ UTR NM_001315.1 2959TTCTCAAAGGGATTCCTACA 158 186908 coding NM_001315.1 678TCTGCCCCCATGAGATGGGT 159 186909 coding NM_001315.1 540TTCGCATGAATGATGGACTG 160 186910 coding NM_001315.1 1275TACTGAGCAAAGTAGGCATG 161 186911 coding NM_001315.1 1336GTCCCTGCTTTCAAAGGACT 162 186912 coding NM_001315.1 577CATATGTTTAAGTAACCGCA 163 186913 3′ UTR NM_001315.1 2963CACATTCTCAAAGGGATTCC 164

[0297] Additional antisense oligonucleotides were designed to targethuman p38α using published sequence (Genbank accession numberNM_(—)001315.1, provided herein as SEQ ID NO: 127. Oligonucleotides weresynthesized as oligonucleotides comprised of 2′-deoxynucleotides andphosphodiester internucleoside linkages (P═O). These oligonucleotidesequences are shown in Table 22. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. TABLE 22 Additional phosphodiester oligonucleotidestargeted to p38α Target SEQ ISIS Sequence Target ID # Region AccessionSite SEQUENCE NO 169107 coding NM_001315.1 1420 GGACTCCATCTCTTCTTGGTCAA165 336747 3′ UTR NM_001315.1 1454 GAAGTGGGATCAACAGAACAGAAA 166 336750coding NM_001315.1 436 AGCCCACTGGAGACAGGTTCT 167

Example 12 Mouse and Rat p38α Antisense Oligonucleotides

[0298] Antisense oligonucleotides were designed to target mouse p38αusing published sequences (Genbank accession number U10871.1, providedherein as SEQ ID NO: 114, GenBank accession number D83073.1, providedherein as SEQ ID NO: 168, GenBank accession number AA002328.1, providedherein as SEQ ID NO: 169, GenBank accession number AF128892.1, providedherein as SEQ ID NO: 170, GenBank accession number BY159314.1, providedherein as SEQ ID NO: 171 and Genbank accession number BY257628.1,provided herein as SEQ ID NO: 172). These compounds are shown in thetables included in this example.

[0299] Antisense oligonucleotides were also designed to target rat p38αusing published sequences (GenBank accession number U73142, providedherein as SEQ ID NO: 45, and Genbank accession number U91847.1, providedherein as SEQ ID NO: 173). These compounds are shown in the tables inthis example.

[0300] Oligonucleotides were synthesized as chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings.” The wings arecomposed of 2′-methoxyethyl (2′-MOE) nucleotides. Internucleosidelinkages are phosphorothioate (P═S). In Table 23, “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds.

[0301] The compounds in Table 23 were analyzed for their effect on mousep38α mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments in which bEND.3cells were treated with the antisense oligonucleotides of the presentinvention and are presented in the column labeled “% inhib, mouse p38α”.If present, “N.D.” indicates “no data”. ISIS 18078(GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 174) was used as a scrambled controloligonucleotide.

[0302] The compounds in Table 23 were also analyzed for their effect onrat p38α mRNA levels in NR-8383 cells by quantitative real-time PCR asdescribed in other examples herein. The rat normal lung alveolarmacrophage cell line NR-8383 was obtained from the American Type CultureCollection (Manassas, Va.). NR-8383 cells were routinely cultured inHam's F12 medium (Gibco/Life Technologies, Gaithersburg, Md.)supplemented with 10% fetal bovine serum (Gibco/Life Technologies,Gaithersburg, Md.), and 1% Penicillin/Streptomycin (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Fortransfection with oligonucleotides, NR-8383 cells were plated on 24 wellplates at a density of 4×10⁴ cells/cm2 (8.0×10⁴ cells/well) inserum-free F12 Nutrient Medium (Gibco/Life Technologies, Gaithersburg,Md.). After 2 hours, media was removed and replaced with 400 ul of Ham'sF12 Nutrient Medium supplemented with 15% fetal bovine serum and 1%Penicillin/Streptomyocin. Cells were then transfected with 300 nM ofantisense oligonucleotides mixed with FuGENE 6

[0303] Transfection Reagent (Roche Applied Science, Indianapolis, Ind.)for 24 hours, after which mRNA was quantitated as described in otherexamples herein. Data are averages from two experiments in which NR-8383cells were treated with the antisense oligonucleotides of the presentinvention and are presented in the column labeled “% inhib, rat p38α”.If present, “N.D.” indicates “no data”. ISIS 18078(GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 174) was used as a scrambled controloligonucleotide.

[0304] One additional compound, ISIS 186911 (SEQ ID NO: 143), targetedto human p38α, was also tested for its effect on mouse and rat p38α mRNAexpression in bEND.3 cells and NR-8383 cells, respectively.

[0305] An asterisk (*) adjacent to the ISIS oligonucleotide number inTable 23 indicates that the oligonucleotide targets human, mouse and ratp38αsequences. Compounds in Table 23, with the exception of ISIS 101753,ISIS 320119, ISIS 320120 and 320121 target both mouse and rat p38α.TABLE 23 Inhibition of mouse and rat p38α by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap Target % Inhib. %Inhib. Sequence Target mouse rat Seq ISIS # Region Accession # SiteSequence p38α p38α ID NO 100864* coding L35253 538 TCCAACAGACCAATCACATT83 57 82 101753 start U73142 1 CTGCGACATTTTCCAGCGGC 64 43 175 codon101755* coding U10871.1 1226 CATCATCAGGGTCGTGGTAC 84 74 176 101757*coding U10871.1 1336 AGGTGCTCAGGACTCCATTT 88 53 177 186911* coding NM001315.1 1336 GTCCCTGCTTTCAAAGGACT 81 40 178 306022* coding U73142 781GGCCAGAGACTGAATGTAGT 78 53 179 320103* coding U10871.1 315AGCTCCTGCCGGTAGAACGT 81 55 180 320104* coding U10871.1 405TCAAAAGCAGCACACACCGA 82 42 181 320105* coding U10871.1 417CCCGTCTTTGTATCAAAAGC 89 59 182 320106* coding U10871.1 453AACGGTCTCGACAGCTTCTT 91 67 183 320107* coding U10871.1 483TAGGTCCTTTTGGCGTGAAT 84 60 184 320108* coding U10871.1 600AGATGGGTCACCAGGTACAC 61 57 185 320109* coding U10871.1 609GCCCCCATGAGATGGGTCAC 69 34 186 320110* coding U10871.1 807TCATCAGTGTGCCGAGCCAG 87 54 187 320111* coding U10871.1 930GTCAACAGCTCAGCCATGAT 86 55 188 320112* coding U10871.1 940CGTTCTTCCGGTCAACAGCT 93 58 189 320113* coding U10871.1 967ATCAATATGGTCTGTACCAG 35 9 190 320114* coding U10871.1 987CTTAAAATGAGCTTCAACTG 71 60 191 320115* coding U10871.1 1001GGGTTCCAACGAGTCTTAAA 67 53 192 320116* coding U10871.1 1019TCAGAAGCTCAGCCCCTGGG 95 73 193 320117* coding U10871.1 1030GGAGATTTTCTTCAGAAGCT 72 55 194 320118* coding U10871.1 1040CAGACTCTGAGGAGATTTTC 47 69 195 320119 coding U10871.1 1050TAGTTTCTTGCAGACTCTGA 53 32 196 320120 coding U10871.1 1060AGACTGAATGTAGTTTCTTG 74 39 197 320121 coding U10871.1 1083TTCATCTTCGGCATCTGGGC 83 57 198 320122 coding U10871.1 1093ATTTGCGAAGTTCATCTTCG 73 48 199 320123 coding U10871.1 1103CAATAAATACATTTGCGAAG 79 32 200 320124 coding U10871.1 1113GGATTGGCACCAATAAATAC 29 31 201 320125 coding U10871.1 1176GCTGCTGTGATCCTCTTATC 67 63 202 320126 coding U10871.1 1196AGGCATGCGCAAGAGCTTGG 90 69 203 320127 coding U10871.1 1206TGAGCAAAGTAGGCATGCGC 73 56 204 320128 coding U10871.1 1260TCAAAGGACTGGTCATAAGG 79 37 205 320129 coding U10871.1 1351CATTTCTTCTTGGTCAAGGG 69 65 206 320130 stop U10871.1 1358AGGACTCCATTTCTTCTTGG 81 61 207 codon 320131 3′ UTR U10871.1 1406CTTCCCCTCACAGTGAAGTG 92 39 208 320132 3′ UTR U10871.1 1432TATTTGGAGAGTTCCCATGA 85 56 209 320133 3′ UTR U10871.1 1442ACTTGAATGGTATTTGGAGA 52 61 210 320134 3′ UTR U10871.1 1452AACAAGAGGCACTTGAATGG 85 74 211 320135 3′ UTR U10871.1 1480ACCCCCTTCCACCATGAAGG 95 47 212 320136 3′ UTR U10871.1 1608AGCAGGCAGACTGCCAAGGA 83 34 213 320137 3′ UTR U10871.1 1663CACACACATCCCTAAGGAGA 80 44 214 320138 3′ UTR U10871.1 1745TAAAGGCAGGGCCACAGGAG 87 46 215 320139 3′ UTR U10871.1 1771GCAGCCTCTCTCTGTCACTG 87 61 216 320140 3′ UTR U10871.1 1791GGGATAGCCTCAGACCTGAA 61 37 217 320141 3′ UTR U10871.1 1801GCATGGCTGAGGGATAGCCT 83 73 218 320142 3′ UTR U10871.1 1828GAGCCAGTTGGTTCTCTTGG 85 53 219 320143 3′ UTR U10871.1 1910AGGCACAAACAGACTGACAG 88 54 220 320144 3′ UTR U10871.1 1917CCTTTTAAGGCACAAACAGA 83 39 221 320145 3′ UTR U10871.1 2138GACCTCTGCACTGAGGTGAA 52 44 222 320146 3′ UTR U10871.1 2147GGCACTGGAGACCTCTGCAC 74 57 223 320147 3′ UTR U10871.1 2228AGAGCACAGCATGCAAACAC 66 43 224 320148 3′ UTR U10871.1 2259CCAGGGCTTCCAGAAGACAG 78 33 225 320149 3′ UTR U10871.1 2576AAGGAGCTCCTGGCTTCAGG 74 25 226 320150 3′ UTR U10871.1 2738GGATTCCTACAACATACAAA 82 62 227 320151 3′ UTR U10871.1 2758GAAGGAACCACACTCTCTAA 90 47 228 320152 3′ UTR U10871.1 2778TTTGCCCTTTCTCCCCATCA 93 66 229 320153 3′ UTR U10871.1 2791AATATTAAAATAATTTGCCC 0 22 230 320154 3′ UTR U10871.1 2817TCATGTTTATAAAGGTGAAA 52 50 231 320155 3′ UTR U10871.1 2827CCCTGAGGATTCATGTTTAT 93 73 232 320156 3′ UTR U10871.1 2930GGAATTGGCTTTACACTTTC 91 64 233 320157 3′ UTR U10871.1 2941CGTCCAACACTGGAATTGGC 96 71 234 320158 3′ UTR U10871.1 3042CCTTCTGGGCTCCAAATGAT 91 71 235 320159 3′ UTR U10871.1 3386TCTGACATCCTATGGCATAC 94 69 236 320160 coding D83073.1 900GTTAATATGGTCTGTACCAG 53 43 237 320161 coding D83073.1 910GCTGAAGCTGGTTAATATGG 80 66 238 320162 coding D83073.1 920CGCATTATCTGCTGAAGCTG 92 62 239 320163 coding D83073.1 955TGTTAATGAGATAAGCAGGG 0 40 240 320164 coding D83073.1 965CTTGGCATCCTGTTAATGAG 80 73 241 320165 coding D83073.1 975TGCCTCATGGCTTGGCATCC 81 53 242 320166 coding D83073.1 991ACTGAATGTAGTTTCTTGCC 53 35 243 320167 5′ UTR AA002328.1 155CTTGCCTGTAAAAACACAGA 7 11 244 320168 stop AF128892.1 1059TCACCTCATGGCTTGGCATC 83 56 245 codon 320169 stop AF128892.1 1066TTTGTTCTCACCTCATGGCT 92 64 246 codon 320170 3′ UTR AF128892.1 1132TGCTGGCTATACACAGACAC 83 55 247 320171 intron BY159314.1 58TGGAAAACTGTTTTGTCAAA 35 2 248 320172 intron 3Y257628.1 39ACTCTCGCGAGAACAGCTCC 39 0 249 320173 intron BY257628.1 72TCCCACAGGCAGCGGCCGGG 160 250 320174 intron BY257628.1 97CCCGCTTGGGCTCCAGTGGC 62 29 251

[0306] Additional antisense oligonucleotides were designed to targetmouse p38α using published sequences (Genbank accession number U10871.1,provided herein as SEQ ID NO: 114). Oligonucleotides are composed of2′-deoxynucleotides. Internucleoside linkages are phosphorodiester(P═O). These oligonucleotide sequences are shown in Table 24. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. TABLE 24 Antisenseoligonucleotides targeted to mouse p38α having 2′-deoxynucleotides andphosphodiester linkages Target Sequence Start SEQ ISIS # RegionAccession # Site SEQUENCE ID NO 137934 3′ UTR U10871.1 3331GCAGTTTTCTCGTTCC 252 CTTG 264006 coding U10871.1 1207 CTGAGCAAAGTAGGCA253 TGCG 320184 3′ UTR U10871.1 2306 GGAGGCAATGTGGACA 254 GGAA 279221coding U10871.1 521 CATTTTCGTGTTTCAT 255 GTGCTTC 326403 3′ UTR U10871.13395 TATTTTAACCAGTGGT 256 ATTATCTACATCCT

[0307] Additional antisense oligonucleotides were designed to targetmouse p38α using published sequences (Genbank accession number U10871.1,provided herein as SEQ ID NO: 114). Oligonucleotides were synthesized aschimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides.Internucleoside linkages in the central gap region are phosphorothioate(P═S), and internucleoside linkages in the wings are phosphodiester(P═O). These oligonucleotide sequences are shown in Table 25. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. TABLE 25 Chimericoligonucleotides targeted to mouse p38a having 2′-MOE wings and a deoxygap and mixed phophorothioate and phosphodiester internucleosidelinkages Target Sequence SEQ ISIS Accession Start ID # Region # SiteSEQUENCE NO 101369 codon U10871.1 286 CTGCGACATCTTCCAGCG 257 GC 101370coding U10871.1 646 GGTCAGCTTCTGGCACTT 258 CA 101372 3′ UTR U10871.11609 AAGCAGGCAGACTGCCAA 259 GG

[0308] Additional antisense oligonucleotides were designed to target ratp38α using published sequences (GenBank accession number U73142,provided herein as SEQ ID NO: 45, and GenBank accession number U91847.1,provided herein as SEQ ID NO: 173). Oligonucleotides are composed of2′-deoxynucleotides. Internucleoside linkages are phosphorodiester(P═O). These oligonucleotide sequences are shown in Table 26. “Targetsite” indicates the first (5′-most) nucleotide number on the particulartarget sequence to which the compound binds. TABLE 26 Antisenseoligonucleotides targeted to rat p38α having 2′-deoxynucleotides andphosphodiester linkages Target Sequence SEQ ISIS Accession Start ID #Region # Site SEQUENCE NO 336744 coding U91847.1 902 AGGCATGCGCAAGAGCTT260 336741 coding U91847.1 66 GGGACAGGTTCTGGTATC 261 GC 257014 codingU91847.1 224 TCTCGTGCTTCATGTGCT 262 TCA 320187 3′ UTR U73142   2800TGGAGCTGGACTGCATAC 263 TGA

[0309] Additional antisense oligonucleotides were designed to target ratp38α using published sequences (GenBank accession number U73142,provided herein as SEQ ID NO: 45). Oligonucleotides were synthesized aschimeric oligonucleotides, composed 2′-deoxynucleotides and2′-methoxyethyl (2′-MOE) nucleotides (indicated in bold type in Table27). Internucleoside linkages in the central gap region arephosphorothioate (P═S), and internucleoside linkages in the wings arephosphodiester (P═O). These oligonucleotide sequences are shown in Table27. “Target site” indicates the first (5′-most) nucleotide number on theparticular target sequence to which the compound binds. TABLE 27Chimeric oligonucleotides targeted to rat p38α having 2′-MOE wings and adeoxy gap and mixed phophorothioate and phosphodiester internucleosidelinkages Target Sequence SEQ ISIS Accession Start ID # Region # SiteSEQUENCE NO 111831 coding U73142 941 CATCAGGGTCGTGGTAC 264 111830 codingU73142 942 CATCATCAGGGTCGT 265

Example 13 Mouse model of allergic inflammation

[0310] In the mouse model of allergic inflammation, mice were sensitizedand challenged with aerosolized chicken ovalbumin (OVA). Airwayresponsiveness was assessed by inducing airflow obstruction with amethacholine aerosol using a noninvasive method. This methodologyutilized unrestrained conscious mice that are placed into the mainchamber of a plthysmograph (Buxco Electronics, Inc., Troy, N.Y.).Pressure differences between this chamber and a reference chamber wereused to extrapolate minute volume, breathing frequency and enhancedpause (Penh). Penh is a dimensionless parameter that is a function oftotal pulmonary airflow in mice (i.e., the sum of the airflow in theupper and lower respiratory tracts) during the respiratory cycle of theanimal. The lower the Penh, the greater the airflow. This parameterclosely correlates with lung resistance as measured by traditionalinvasive techniques using ventilated animals (Hamelmann et al., 1997).Dose-response data were plotted as raw Penh values to increasingconcentrations of methacholine. This system was used to test theefficacy of an antisense oligonucleotide targeted to mouse p38α (ISIS101757; SEQ ID NO: 177). Mismatched p38α oligonucleotide (ISIS 101758;SEQ ID NO: 266) was used as a negative control.

[0311] There are several important features common to human asthma andthe mouse model of allergic inflammation. One of these is pulmonaryinflammation, in which cytokine expression and Th2 profile is dominant.Another is goblet cell hyperplasia with increased mucus production.Lastly, airway hyperresponsiveness (AHR) occurs resulting in increasedsensitivity to cholinergic receptor agonists such as acetylcholine ormethacholine. The compositions and methods of the present invention maybe used to treat AHR and pulmonary inflammation. The combined use ofantisense oligonucleotides targeted to human p38 MAP kinase with one ormore conventional asthma medications including, but not limited to,montelukast sodium (Singulair™), albuterol, beclomethasone dipropionate,triamcinolone acetonide, ipratropium bromide (Atrovent™), flunisolide,fluticasone propionate (Flovent™) and other steroids is alsocontemplated.

[0312] Ovalbumin-Induced Allergic Inflammation

[0313] For intratracheal administration of ISIS 101757, female Balb/cmice (Charles Rivers Laboratory, Taconic Farms, N.Y.) were maintained inmicro-isolator cages housed in a specific pathogen-free (SPF) facility.The sentinel cages within the animal colony surveyed negative for viralantibodies and the presence of known mouse pathogens. Mice weresensitized and challenged with aerosolized chicken OVA. Briefly, 20 μmalum-precipitated OVA was injected intraperitoneally on days 0 and 14.On day 24, 25 and 26, the animals were exposed for 20 minutes to 1.0%OVA (in saline) by nebulization. The challenge was conducted using anultrasonic nebulizer (PulmoSonic, The DeVilbiss Co., Somerset, Pa.).Animals were analyzed about 24 hours following the last nebulizationusing the Buxco electronics Biosystem. Lung function (Penh), lunghistology (cell infiltration and mucus production), target mRNAreduction in the lung, inflammation (BAL cell type & number, cytokinelevels), spleen weight and serum AST/ALT were determined.

[0314] For the aerosol studies, the protocol described above wasslightly modified. Male Balb/c mice were injected IP with OVA (20 μg) inaluminum hydroxide on days 0 and 14. Aerosol dosing was performed withnebulized sterile saline, antisense oligonucleotide or mismatchedcontrol oligonucleotide using 25, 125 and 250 μg/ml solutions (5 mg/kg)for 30 min. on days 14-20 in a closed chamber. Aerosol lung challengewas carried out with nebulized saline or 1% OVA for 20 min. on days 18,19 and 20. BAL fluid was collected at 24 hr post-last lung challenge(cell differentials) or at 2-12 h post-challenge (cytokine analysis).AHR was measured 24 hours after OVA challenge. Mice were exposed toaerosolized methacholine 24 hr post-last lung challenge from 2-80 mg/mlfor 3 min. until a 200% increase in Penh was achieved.

[0315] Intratracheal Oligonucleotide Administration

[0316] Antisense oligonucleotides (ASOs) were dissolved in saline andused to intratracheally dose mice every day, four times per day, fromdays 15-26 of the OVA sensitization and challenge protocol, or used asan aerosol. Specifically, the mice were anesthetized with isofluoraneand placed on a board with the front teeth hung from a line. The nosewas covered and the animal's tongue was extended with forceps and 25 μlof various doses of ASO, or an equivalent volume of saline (control) wasplaced at the back of the tongue until inhaled into the lung.

[0317] Mouse antisense oligonucleotides to p38α are phosphorothioateswith 2′-MOE modifications on nucleotides 1-5 and 16-20, and 2′-deoxy atpositions 6-15. These ASOs were identified by mouse-targeted ASOscreening of 10 p38α antisense oligonucleotides by target p38α mRNAreduction in mouse bEND.3 cells, as described in Example 12.Dose-response confirmation led to selection of ISIS 21873 (>70%reduction at 50 nM). ISIS 101757 contains all phosphorothioate linkages,whereas ISIS 21873 is a mixed phosphodiester/phosphorothioate compound.ISIS 101757 had an IC50<50 nM for reducing p38α mRNA in endothelialcells, and an IC50 of about 250 nM in fibroblasts.

[0318] Results of Aerosol Administration

[0319] The p38α knock-down effect of ISIS 101757 was confirmed in amouse T cell line (EL4) and a mouse macrophage cell line (RAW264.7)using Western blotting. ISIS 101757, but not the mismatched control,dose-dependently suppressed methacholine-induced AHR in sensitized micemeasured by whole body plethysmography (FIG. 1A-1B). The PC200 valuesfor methacholine (FIG. 2) significantly (P<0.05) reduced OVA-inducedincreases in total cell counts and eosinophils recovered in BAL fluid(FIG. 3). In addition, histological studies revealed that ISIS 101757markedly inhibited OVA-induced inflammatory cell infiltration into thelungs (H&E stain) and mucus hypersecretion in the airway epithelium (PASstain). ISIS 101757 also significantly (P<0.05);lowered blood levels oftotal IgE, OVA-specific IgE and OVA-specific IgG₁ in sensitized mice ascompared to the mismatched control. Oligonucleotide levels of up to 1μg/g of lung tissue were sufficient to achieve the pharmacologicaleffects described above. The aerosolized ISIS 101757 concentration inmouse lung vs. dose is shown in FIG. 4. There was no significant effectof aerosol oligonucleotide administration of spleen weight. These dataindicate that p38α antisense oligonucleotides are useful for thetreatment of asthma.

[0320] Intratracheal Administration Results

[0321] After intratracheal administration of ISIS 101757 as describedabove, dose-dependent inhibition of the Penh response to methacholine(50 mg/ml) challenge was observed (FIG. 5). The oligonucleotideconcentration (μg/g) in lungs vs. dose is shown in FIG. 6.

[0322] RT-PCR Analysis

[0323] RNA was harvested from experimental lungs removed on day 28 ofthe OVA protocol. P38α levels were measured by quantitative RT-PCR asdescribed in other examples herein.

[0324] Collection of Bronchial Alveolar Lavage (BAL) Fluid and BloodSerum for the Determination of Cytokine and Chemokine Levels

[0325] Animals were injected with a lethal dose of ketamine, the tracheawas exposed and a cannula was inserted and secured by sutures. The lungswere lavaged twice with 0.5 ml aliquots of ice cold PBS with 0.2% FCS.The recovered BAL fluid was centrifuged at 1,000 rpm for 10 min at 4°C., frozen on dry ice and stored at −80° C. until used. Luminex was usedto measure cytokine levels in BAL fluid and serum.

[0326] BAL Cell Counts and Differentials

[0327] Cytospins of cells recovered from BAL fluid were prepared using aShandon Cytospin 3 (Shandon Scientific LTD, Cheshire, England). Celldifferentials were performed from slides stained with Leukostat (FisherScientific, Pittsburgh, Pa.). Total cell counts were quantified byhemocytometer and, together with the percent type by differential, wereused to calculate specific cell number.

[0328] Tissue Histology

[0329] Before resection, lungs were inflated with 0.5 ml of 10%phosphate-buffered formalin and fixed overnight at 4° C. The lungsamples were washed free of formalin with 1×PBS and subsequentlydehydrated through an ethanol series prior to equilibration in xyleneand embedded in paraffin. Sections (6μ) were mounted on slides andstained with hematoxylin/eosin, massons trichome and periodicacid-schiff (PAS) reagent. Parasagittal sections were analyzed bybright-field microscopy. Mucus cell content was assessed as the airwayepithelium staining with PAS. Relative comparisons of mucus content weremade between cohorts of animals by counting the number of PAS-positiveairways.

Example 14 Design and Screening of Duplexed Antisense CompoundsTargeting p38α MAP Kinase

[0330] In accordance with the present invention, a series of nucleicacid duplexes comprising the antisense compounds of the presentinvention and their complements can be designed to target p38α MAPkinase. The nucleobase sequence of the antisense strand of the duplexcomprises at least a portion of an oligonucleotide to p38α MAP kinase asdescribed herein. The ends of the strands may be modified by theaddition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the dsRNA is then designed and synthesizedas the complement of the antisense strand and may also containmodifications or additions to either terminus. For example, in oneembodiment, both strands of the dsRNA duplex would be complementary overthe central nucleobases, each having overhangs at one or both termini.For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:  cgagaggcggacgggaccgTT Antisense Strand   |||||||||||||||||||TTgctctccgcctgccctggc Complement

[0331] RNA strands of the duplex can be synthesized by methods disclosedherein or purchased from Dharmacon Research Inc., (Lafayette, Colo.).Once synthesized, the complementary strands are annealed. The singlestrands are aliquoted and diluted to a concentration of 50 uM. Oncediluted, 30 uL of each strand is combined with 15 uL of a 5×solution ofannealing buffer. The final concentration of said buffer is 100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate.The final volume is 75 uL. This solution is incubated for 1 minute at90° C. and then centrifuged for 15 seconds. The tube is allowed to sitfor 1 hour at 37° C. at which time the dsRNA duplexes are used inexperimentation. The final concentration of the dsRNA duplex is 20 uM.This solution can be stored frozen (−20° C.) and freeze-thawed up to 5times.

[0332] Once prepared, the duplexed antisense compounds are evaluated fortheir ability to modulate p38α MAP kinase expression according to theprotocols described herein.

Example 15 Design of Phenotypic Assays and In Vivo Studies for the Useof p38α MAP Kinase Inhibitors

[0333] Phenotypic Assays

[0334] Once p38α MAP kinase inhibitors have been identified by themethods disclosed herein, the compounds are further investigated in oneor more phenotypic assays, each having measurable endpoints predictiveof efficacy in the treatment of a particular disease state or condition.Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of p38α MAP kinase in health and disease.Representative phenotypic assays, which can be purchased from any one ofseveral commercial vendors, include those for determining cellviability, cytotoxicity, proliferation or cell survival (MolecularProbes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assaysincluding enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences,Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.),cell regulation, signal transduction, inflammation, oxidative processesand apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

[0335] In one non-limiting example, cells determined to be appropriatefor a particular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with p38αMAP kinase inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

[0336] Phenotypic endpoints include changes in cell morphology over timeor treatment dose as well as changes in levels of cellular componentssuch as proteins, lipids, nucleic acids, hormones, saccharides ormetals. Measurements of cellular status which include pH, stage of thecell cycle, intake or excretion of biological indicators by the cell,are also endpoints of interest.

[0337] Analysis of the genotype of the cell (measurement of theexpression of one or more of the genes of the cell) after treatment isalso used as an indicator of the efficacy or potency of the p38α MAPkinase inhibitors. Hallmark genes, or those genes suspected to beassociated with a specific disease state, condition, or phenotype, aremeasured in both treated and untreated cells.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 266 <210> SEQ ID NO 1<211> LENGTH: 1539 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (295)..(1377) <300>PUBLICATION INFORMATION: <303> JOURNAL: Science <304> VOLUME: 265 <305>ISSUE: 5173 <306> PAGES: 808-811 <307> DATE: 1994-08-05 <308> DATABASEACCESSION NUMBER: L35253 <309> DATABASE ENTRY DATE: 1995-08-14 <400>SEQUENCE: 1 ggaattccgg gcccggtctt tcctcccgcc gccgccggcc tggtcccggggactggcctc 60 cacgtccgac tcgtccgagc tgaagcccag cagcactttg ctgccagccgcgggggcggc 120 ggaggcgccc ccgggccctc ccaggaggct ctctgggcca gaggccgagattcggcacag 180 gcccccagga gtccgtaagt aggagaggtc gcccgagacc ggccggacccccatccccgc 240 ggccgccgcc gccgctggtc ccgcggctgc gaccgtggcg gctgccgctggaaa atg 297 Met 1 tct cag gag agg ccc acg ttc tac cgg cag gag ctg aacaag aca atc 345 Ser Gln Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn LysThr Ile 5 10 15 tgg gag gtg ccc gag cgt tac cag aac ctg tct cca gtg ggctct ggc 393 Trp Glu Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly SerGly 20 25 30 gcc tat ggc tct gtg tgt gct gct ttt gac aca aaa acg ggg ttacgt 441 Ala Tyr Gly Ser Val Cys Ala Ala Phe Asp Thr Lys Thr Gly Leu Arg35 40 45 gtg gca gtg aag aag ctc tcc aga cca ttt cag tcc atc att cat gcg489 Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile His Ala 5055 60 65 aaa aga acc tac aga gaa ctg cgg tta ctt aaa cat atg aaa cat gaa537 Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His Glu 7075 80 aat gtg att ggt ctg ttg gac gtt ttt aca cct gca agg tct ctg gag585 Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu Glu 8590 95 gaa ttc aat gat gtg tat ctg gtg acc cat ctc atg ggg gca gat ctg633 Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp Leu 100105 110 aac aac att gtg aaa tgt cag aag ctt aca gat gac cat gtt cag ttc681 Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln Phe 115120 125 ctt atc tac caa att ctc cga ggt cta aag tat ata cat tca gct gac729 Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asp 130135 140 145 ata att cac agg gac cta aaa cct agt aat cta gct gtg aat gaagac 777 Ile Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu Asp150 155 160 tgt gag ctg aag att ctg gat ttt gga ctg gct cgg cac aca gatgat 825 Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp Asp165 170 175 gaa atg aca ggc tac gtg gcc act agg tgg tac agg gct cct gagatc 873 Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile180 185 190 atg ctg aac tgg atg cat tac aac cag aca gtt gat att tgg tcagtg 921 Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser Val195 200 205 gga tgc ata atg gcc gag ctg ttg act gga aga aca ttg ttt cctggt 969 Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro Gly210 215 220 225 aca gac cat att gat cag ttg aag ctc att tta aga ctc gttgga acc 1017 Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu Arg Leu Val GlyThr 230 235 240 cca ggg gct gag ctt ttg aag aaa atc tcc tca gag tct gcaaga aac 1065 Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser Ala ArgAsn 245 250 255 tat att cag tct ttg act cag atg ccg aag atg aac ttt gcgaat gta 1113 Tyr Ile Gln Ser Leu Thr Gln Met Pro Lys Met Asn Phe Ala AsnVal 260 265 270 ttt att ggt gcc aat ccc ctg gct gtc gac ttg ctg gag aagatg ctt 1161 Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu Leu Glu Lys MetLeu 275 280 285 gta ttg gac tca gat aag aga att aca gcg gcc caa gcc cttgca cat 1209 Val Leu Asp Ser Asp Lys Arg Ile Thr Ala Ala Gln Ala Leu AlaHis 290 295 300 305 gcc tac ttt gct cag tac cac gat cct gat gat gaa ccagtg gcc gat 1257 Ala Tyr Phe Ala Gln Tyr His Asp Pro Asp Asp Glu Pro ValAla Asp 310 315 320 cct tat gat cag tcc ttt gaa agc agg gac ctc ctt atagat gag tgg 1305 Pro Tyr Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu Ile AspGlu Trp 325 330 335 aaa agc ctg acc tat gat gaa gtc atc agc ttt gtg ccacca ccc ctt 1353 Lys Ser Leu Thr Tyr Asp Glu Val Ile Ser Phe Val Pro ProPro Leu 340 345 350 gac caa gaa gag atg gag tcc tga gcacctggtttctgttctgt tgatcccact 1407 Asp Gln Glu Glu Met Glu Ser 355 360tcactgtgag gggaaggcct tttcacggga actctccaaa tattattcaa gtgcctcttg 1467ttgcagagat ttcctccatg gtggaagggg gtgtgcgtgc gtgtgcgtgc gtgttagtgt 1527gtgtgcatgt gt 1539 <210> SEQ ID NO 2 <211> LENGTH: 360 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Ser Gln Glu Arg ProThr Phe Tyr Arg Gln Glu Leu Asn Lys Thr 1 5 10 15 Ile Trp Glu Val ProGlu Arg Tyr Gln Asn Leu Ser Pro Val Gly Ser 20 25 30 Gly Ala Tyr Gly SerVal Cys Ala Ala Phe Asp Thr Lys Thr Gly Leu 35 40 45 Arg Val Ala Val LysLys Leu Ser Arg Pro Phe Gln Ser Ile Ile His 50 55 60 Ala Lys Arg Thr TyrArg Glu Leu Arg Leu Leu Lys His Met Lys His 65 70 75 80 Glu Asn Val IleGly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu 85 90 95 Glu Glu Phe AsnAsp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp 100 105 110 Leu Asn AsnIle Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln 115 120 125 Phe LeuIle Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala 130 135 140 AspIle Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu 145 150 155160 Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp 165170 175 Asp Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu180 185 190 Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile TrpSer 195 200 205 Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr LeuPhe Pro 210 215 220 Gly Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu ArgLeu Val Gly 225 230 235 240 Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile SerSer Glu Ser Ala Arg 245 250 255 Asn Tyr Ile Gln Ser Leu Thr Gln Met ProLys Met Asn Phe Ala Asn 260 265 270 Val Phe Ile Gly Ala Asn Pro Leu AlaVal Asp Leu Leu Glu Lys Met 275 280 285 Leu Val Leu Asp Ser Asp Lys ArgIle Thr Ala Ala Gln Ala Leu Ala 290 295 300 His Ala Tyr Phe Ala Gln TyrHis Asp Pro Asp Asp Glu Pro Val Ala 305 310 315 320 Asp Pro Tyr Asp GlnSer Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu 325 330 335 Trp Lys Ser LeuThr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro 340 345 350 Leu Asp GlnGlu Glu Met Glu Ser 355 360 <210> SEQ ID NO 3 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 3 aagaccgggc ccggaattcc20 <210> SEQ ID NO 4 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 4 gtggaggcca gtccccggga ccggaattcc 30 <210> SEQID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: antisense sequence<400> SEQUENCE: 5 tggcagcaaa gtgctgctgg 20 <210> SEQ ID NO 6 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 6cagagagcct cctgggaggg 20 <210> SEQ ID NO 7 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 7 tgtgccgaat ctcggcctct20 <210> SEQ ID NO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 8 ggtctcgggc gacctctcct 20 <210> SEQ ID NO 9<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 9 cagccgcggg accagcggcg 20 <210> SEQ ID NO 10 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: antisense sequence <400> SEQUENCE: 10 cattttccagcggcagccgc 20 <210> SEQ ID NO 11 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 11 tcctgagaca ttttccagcg 20 <210> SEQID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: antisense sequence<400> SEQUENCE: 12 ctgccggtag aacgtgggcc 20 <210> SEQ ID NO 13 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 13gtaagcttct gacatttcac 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 14 tttaggtccc tgtgaattat20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 15 atgttcttcc agtcaacagc 20 <210> SEQ ID NO 16<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 16 taaggaggtc cctgctttca 20 <210> SEQ ID NO 17 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 17aaccaggtgc tcaggactcc 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 18 gaagtgggat caacagaaca20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 19 tgaaaaggcc ttcccctcac 20 <210> SEQ ID NO 20<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 20 aggcacttga ataatatttg 20 <210> SEQ ID NO 21 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 21cttccaccat ggaggaaatc 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 22 acacatgcac acacactaac20 <210> SEQ ID NO 23 <211> LENGTH: 2180 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(20)..(1138) <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: U53442 <309> DATABASE ENTRY DATE: 1996-07-30 <400> SEQUENCE: 23gtgaaattct gctccggac atg tcg ggc cct cgc gcc ggc ttc tac cgg cag 52 MetSer Gly Pro Arg Ala Gly Phe Tyr Arg Gln 1 5 10 gag ctg aac aag acc gtgtgg gag gtg ccg cag cgg ctg cag ggg ctg 100 Glu Leu Asn Lys Thr Val TrpGlu Val Pro Gln Arg Leu Gln Gly Leu 15 20 25 cgc ccg gtg ggc tcc ggc gcctac ggc tcc gtc tgt tcg gcc tac gac 148 Arg Pro Val Gly Ser Gly Ala TyrGly Ser Val Cys Ser Ala Tyr Asp 30 35 40 gcc cgg ctg cgc cag aag gtg gcggtg aag aag ctg tcg cgc ccc ttc 196 Ala Arg Leu Arg Gln Lys Val Ala ValLys Lys Leu Ser Arg Pro Phe 45 50 55 cag tcg ctg atc cac gcg cgc aga acgtac cgg gag ctg cgg ctg ctc 244 Gln Ser Leu Ile His Ala Arg Arg Thr TyrArg Glu Leu Arg Leu Leu 60 65 70 75 aag cac ctg aag cac gag aac gtc atcggg ctt ctg gac gtc ttc acg 292 Lys His Leu Lys His Glu Asn Val Ile GlyLeu Leu Asp Val Phe Thr 80 85 90 ccg gcc acg tcc atc gag gac ttc agc gaagtg tac ttg gtg acc acc 340 Pro Ala Thr Ser Ile Glu Asp Phe Ser Glu ValTyr Leu Val Thr Thr 95 100 105 ctg atg ggc gcc gac ctg aac aac atc gtcaag tgc cag gcg ggc gcc 388 Leu Met Gly Ala Asp Leu Asn Asn Ile Val LysCys Gln Ala Gly Ala 110 115 120 cat cag ggt gcc cgc ctg gca ctt gac gagcac gtt caa ttc ctg gtt 436 His Gln Gly Ala Arg Leu Ala Leu Asp Glu HisVal Gln Phe Leu Val 125 130 135 tac cag ctg ctg cgc ggg ctg aag tac atccac tcg gcc ggg atc atc 484 Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile HisSer Ala Gly Ile Ile 140 145 150 155 cac cgg gac ctg aag ccc agc aac gtggct gtg aac gag gac tgt gag 532 His Arg Asp Leu Lys Pro Ser Asn Val AlaVal Asn Glu Asp Cys Glu 160 165 170 ctc agg atc ctg gat ttc ggg ctg gcgcgc cag gcg gac gag gag atg 580 Leu Arg Ile Leu Asp Phe Gly Leu Ala ArgGln Ala Asp Glu Glu Met 175 180 185 acc ggc tat gtg gcc acg cgc tgg taccgg gca cct gag atc atg ctc 628 Thr Gly Tyr Val Ala Thr Arg Trp Tyr ArgAla Pro Glu Ile Met Leu 190 195 200 aac tgg atg cat tac aac caa aca gtggat atc tgg tcc gtg ggc tgc 676 Asn Trp Met His Tyr Asn Gln Thr Val AspIle Trp Ser Val Gly Cys 205 210 215 atc atg gct gag ctg ctc cag ggc aaggcc ctc ttc ccg gga agc gac 724 Ile Met Ala Glu Leu Leu Gln Gly Lys AlaLeu Phe Pro Gly Ser Asp 220 225 230 235 tac att gac cag ctg aag cgc atcatg gaa gtg gtg ggc aca ccc agc 772 Tyr Ile Asp Gln Leu Lys Arg Ile MetGlu Val Val Gly Thr Pro Ser 240 245 250 cct gag gtt ctg gca aaa atc tcctcg gaa cac gcc cgg aca tat atc 820 Pro Glu Val Leu Ala Lys Ile Ser SerGlu His Ala Arg Thr Tyr Ile 255 260 265 cag tcc ctg ccc ccc atg ccc cagaag gac ctg agc agc atc ttc cgt 868 Gln Ser Leu Pro Pro Met Pro Gln LysAsp Leu Ser Ser Ile Phe Arg 270 275 280 gga gcc aac ccc ctg gcc ata gacctc ctt gga agg atg ctg gtg ctg 916 Gly Ala Asn Pro Leu Ala Ile Asp LeuLeu Gly Arg Met Leu Val Leu 285 290 295 gac agt gac cag agg gtc agt gcagct gag gca ctg gcc cac gcc tac 964 Asp Ser Asp Gln Arg Val Ser Ala AlaGlu Ala Leu Ala His Ala Tyr 300 305 310 315 ttc agc cag tac cac gac cccgag gat gag cca gag gcc gag cca tat 1012 Phe Ser Gln Tyr His Asp Pro GluAsp Glu Pro Glu Ala Glu Pro Tyr 320 325 330 gat gag agc gtt gag gcc aaggag cgc acg ctg gag gag tgg aag gag 1060 Asp Glu Ser Val Glu Ala Lys GluArg Thr Leu Glu Glu Trp Lys Glu 335 340 345 ctc act tac cag gaa gtc cttagc ttc aag ccc cca gag cca ccg aag 1108 Leu Thr Tyr Gln Glu Val Leu SerPhe Lys Pro Pro Glu Pro Pro Lys 350 355 360 cca cct ggc agc ctg gag attgag cag tga ggtgctgccc agcagcccct 1158 Pro Pro Gly Ser Leu Glu Ile GluGln 365 370 gagagcctgt ggaggggctt gggcctgcac ccttccacag ctggcctggtttcctcgaga 1218 ggcacctccc acactcctat ggtcacagac ttctggccta ggacccctcgccttcaggag 1278 aatctacacg catgtatgca tgcacaaaca tgtgtgtaca tgtgcttgccatgtgtagga 1338 gtctgggcac aagtgtccct gggcctacct tggtcctcct gtcctcttctggctactgca 1398 ctctccactg ggacctgact gtggggtcct agatgccaaa ggggttcccctgcggagttc 1458 ccctgtctgt cccaggccga cccaagggag tgtcagcctt gggctctcttctgtcccagg 1518 gctttctgga gggcgcgctg gggccgggac cccgggagac tcaaagggagaggtctcagt 1578 ggttagagct gctcagcctg gaggtagggc gctgtcttgg tcactgctgagacccacagg 1638 tctaagagga gaggcagagc cagtgtgcca ccaggctggg cagggacaaccaccaggtgt 1698 caaatgagaa aagctgcctg gagtcttgtg ttcacccgtg ggtgtgtgtgggcacgtgtg 1758 gatgagcgtg cactccccgt gttcatatgt cagggcacat gtgatgtggtgcgtgtgaat 1818 ctgtgggcgc ccaaggccag cagccatatc tggcaagaag ctggagccggggtgggtgtg 1878 ctgttgcctt ccctctcctc ggttcctgat gccttgaggg gtgtttcagactggcggcac 1938 cgttgtggcc ctgcagccgg agatctgagg tgctctggtc tgtgggtcagtcctctttcc 1998 ttgtcccagg atggagctga tccagtaacc tcggagacgg gaccctgcccagagctgagt 2058 tgggggtgtg gctctgccct ggaaaggggg tgacctcttg cctcgaggggcccagggaag 2118 cctgggtgtc aagtgcctgc accaggggtg cacaataaag ggggttctctctcagaaaaa 2178 aa 2180 <210> SEQ ID NO 24 <211> LENGTH: 372 <212> TYPE:PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Met Ser Gly Pro ArgAla Gly Phe Tyr Arg Gln Glu Leu Asn Lys Thr 1 5 10 15 Val Trp Glu ValPro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser 20 25 30 Gly Ala Tyr GlySer Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln 35 40 45 Lys Val Ala ValLys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His 50 55 60 Ala Arg Arg ThrTyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His 65 70 75 80 Glu Asn ValIle Gly Leu Leu Asp Val Phe Thr Pro Ala Thr Ser Ile 85 90 95 Glu Asp PheSer Glu Val Tyr Leu Val Thr Thr Leu Met Gly Ala Asp 100 105 110 Leu AsnAsn Ile Val Lys Cys Gln Ala Gly Ala His Gln Gly Ala Arg 115 120 125 LeuAla Leu Asp Glu His Val Gln Phe Leu Val Tyr Gln Leu Leu Arg 130 135 140Gly Leu Lys Tyr Ile His Ser Ala Gly Ile Ile His Arg Asp Leu Lys 145 150155 160 Pro Ser Asn Val Ala Val Asn Glu Asp Cys Glu Leu Arg Ile Leu Asp165 170 175 Phe Gly Leu Ala Arg Gln Ala Asp Glu Glu Met Thr Gly Tyr ValAla 180 185 190 Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Trp MetHis Tyr 195 200 205 Asn Gln Thr Val Asp Ile Trp Ser Val Gly Cys Ile MetAla Glu Leu 210 215 220 Leu Gln Gly Lys Ala Leu Phe Pro Gly Ser Asp TyrIle Asp Gln Leu 225 230 235 240 Lys Arg Ile Met Glu Val Val Gly Thr ProSer Pro Glu Val Leu Ala 245 250 255 Lys Ile Ser Ser Glu His Ala Arg ThrTyr Ile Gln Ser Leu Pro Pro 260 265 270 Met Pro Gln Lys Asp Leu Ser SerIle Phe Arg Gly Ala Asn Pro Leu 275 280 285 Ala Ile Asp Leu Leu Gly ArgMet Leu Val Leu Asp Ser Asp Gln Arg 290 295 300 Val Ser Ala Ala Glu AlaLeu Ala His Ala Tyr Phe Ser Gln Tyr His 305 310 315 320 Asp Pro Glu AspGlu Pro Glu Ala Glu Pro Tyr Asp Glu Ser Val Glu 325 330 335 Ala Lys GluArg Thr Leu Glu Glu Trp Lys Glu Leu Thr Tyr Gln Glu 340 345 350 Val LeuSer Phe Lys Pro Pro Glu Pro Pro Lys Pro Pro Gly Ser Leu 355 360 365 GluIle Glu Gln 370 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 25 cgacatgtcc ggagcagaat20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 26 ttcagctcct gccggtagaa 20 <210> SEQ ID NO 27<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 27 tgcggcacct cccacacggt 20 <210> SEQ ID NO 28 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 28ccgaacagac ggagccgtat 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 29 gtgcttcagg tgcttgagca20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 30 gcgtgaagac gtccagaagc 20 <210> SEQ ID NO 31<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 31 acttgacgat gttgttcagg 20 <210> SEQ ID NO 32 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 32aacgtgctcg tcaagtgcca 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 33 atcctgagct cacagtcctc20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 34 actgtttggt tgtaatgcat 20 <210> SEQ ID NO 35<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 35 atgatgcgct tcagctggtc 20 <210> SEQ ID NO 36 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 36gccagtgcct cagctgcact 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 37 aacgctctca tcatatggct20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 38 cagcacctca ctgctcaatc 20 <210> SEQ ID NO 39<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 39 tctgtgacca taggagtgtg 20 <210> SEQ ID NO 40 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 40acacatgttt gtgcatgcat 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 41 cctacacatg gcaagcacat20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 42 tccaggctga gcagctctaa 20 <210> SEQ ID NO 43<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 43 agtgcacgct catccacacg 20 <210> SEQ ID NO 44 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 44cttgccagat atggctgctg 20 <210> SEQ ID NO 45 <211> LENGTH: 3132 <212>TYPE: DNA <213> ORGANISM: Rattus norvegicus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (12)..(1094) <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: U73142 <309> DATABASEENTRY DATE: 1996-10-22 <400> SEQUENCE: 45 gccgctggaa a atg tcg cag gaaagg ccc acg ttc tac cgg cag gag ctg 50 Met Ser Gln Glu Arg Pro Thr PheTyr Arg Gln Glu Leu 1 5 10 aac aag acc gtc tgg gag gtg ccc gag cga taccag aac ctg tcc ccg 98 Asn Lys Thr Val Trp Glu Val Pro Glu Arg Tyr GlnAsn Leu Ser Pro 15 20 25 gtg ggc tcg gga gcc tac ggc tcg gtg tgt gct gctttt gat aca aag 146 Val Gly Ser Gly Ala Tyr Gly Ser Val Cys Ala Ala PheAsp Thr Lys 30 35 40 45 acg gga cat cgt gtg gca gtg aag aag ctg tcg agaccg ttt cag tcc 194 Thr Gly His Arg Val Ala Val Lys Lys Leu Ser Arg ProPhe Gln Ser 50 55 60 atc att cac gcc aaa agg acc tac agg gag ctg cgg ctgctg aag cac 242 Ile Ile His Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu LeuLys His 65 70 75 atg aag cac gag aat gtg att ggt ctg ttg gat gtg ttt acacct gca 290 Met Lys His Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr ProAla 80 85 90 agg tcc ctg gaa gaa ttc aac gat gtg tac ctg gtg acc cat ctcatg 338 Arg Ser Leu Glu Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met95 100 105 ggg gca gac ctg aac aac atc gtg aag tgt cag aag ctt acc gatgac 386 Gly Ala Asp Leu Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp110 115 120 125 cac gtt cag ttt ctt atc tac cag atc ctg cga ggg ctg aagtat ata 434 His Val Gln Phe Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys TyrIle 130 135 140 cac tcg gct gac ata atc cac agg gac cta aag ccc agc aacctc gct 482 His Ser Ala Asp Ile Ile His Arg Asp Leu Lys Pro Ser Asn LeuAla 145 150 155 gtg aat gaa gac tgt gag ctg aag att ctg gat ttt ggg ctggct cgg 530 Val Asn Glu Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu AlaArg 160 165 170 cac act gat gac gaa atg acc ggc tac gtg gct acc cgg tggtac aga 578 His Thr Asp Asp Glu Met Thr Gly Tyr Val Ala Thr Arg Trp TyrArg 175 180 185 gcc ccc gag att atg ctg aat tgg atg cac tac aac cag acagtg gat 626 Ala Pro Glu Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr ValAsp 190 195 200 205 att tgg tcc gtg ggc tgc atc atg gct gag ctg ttg accgga aga acg 674 Ile Trp Ser Val Gly Cys Ile Met Ala Glu Leu Leu Thr GlyArg Thr 210 215 220 ttg ttt cct ggt aca gac cat att gat cag ttg aag ctcatt tta aga 722 Leu Phe Pro Gly Thr Asp His Ile Asp Gln Leu Lys Leu IleLeu Arg 225 230 235 ctc gtt gga acc cca ggg gct gag ctt ctg aag aaa atctcc tca gag 770 Leu Val Gly Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile SerSer Glu 240 245 250 tct gca aga aac tac att cag tct ctg gcc cag atg ccgaag atg aac 818 Ser Ala Arg Asn Tyr Ile Gln Ser Leu Ala Gln Met Pro LysMet Asn 255 260 265 ttc gca aat gta ttt att ggt gcc aat ccc ctg gct gtcgac ctg ctg 866 Phe Ala Asn Val Phe Ile Gly Ala Asn Pro Leu Ala Val AspLeu Leu 270 275 280 285 gaa aag atg ctg gtt ttg gac tcg gat aag agg atcaca gca gcc caa 914 Glu Lys Met Leu Val Leu Asp Ser Asp Lys Arg Ile ThrAla Ala Gln 290 295 300 gct ctt gcg cat gcc tac ttt gct cag tac cac gaccct gat gat gag 962 Ala Leu Ala His Ala Tyr Phe Ala Gln Tyr His Asp ProAsp Asp Glu 305 310 315 cca gtg gct gaa cct tat gac cag tcc ttt gaa agcagg gac ttc ctt 1010 Pro Val Ala Glu Pro Tyr Asp Gln Ser Phe Glu Ser ArgAsp Phe Leu 320 325 330 ata gac gaa tgg aag agc ctg acc tac gat gaa gtcatt agc ttt gtg 1058 Ile Asp Glu Trp Lys Ser Leu Thr Tyr Asp Glu Val IleSer Phe Val 335 340 345 cca ccg ccc ctt gac caa gaa gaa atg gag tcc tgagcaccttgct 1104 Pro Pro Pro Leu Asp Gln Glu Glu Met Glu Ser 350 355 360tctgttctgt ccatcccact tcactgtgag gggaaggcct gttcatggga actctccaaa 1164taccattcaa gtgcctcttg ttgaaagatt ccttcatggt ggaagggggt gcatgtatgt 1224gcgtagtgtt tgtgtgtgtc tgtctgtctg tccgtttgtc catgtatctt tgtggaagtc 1284attgtgatgg cagtgacttc atgagtggta gatgctcctt ggcagtctgc ctgctctctc 1344agagtccggg caggccgatg ggaactgccg tctccttagg gatgtgtgtg tgtatgttaa 1404gtgcaaagta agaatattaa aatatccctg ttcctagtta ccttgccact tcggcttctc 1464ctgtggccct gcctttacca tatcacagtg acagagagag gctgcttcag gtctgaggct 1524atccctcagc catgcataaa gcccaagaga accaactggc tcctgggctc tagcctgtga 1584tcggcttgct catgtcctca gaacctgtca gtctgtttgt gccttaaaag gagagaaggg 1644cgcgttgtgg tagttacaga atctcagttg ctggcgttct gagccaggca aggcacaggg 1704ctgttggatg gccagtgggg agctggacaa aacaaggcag ccttcaagga ggccatgggt 1764gcatgtttgc atgagtgtat gtgcaaccgc cctccctcac ctccaggagc aagctgtttt 1824ctatgcttac ctaagttcac ctcagtgcag aggtctccag tgccaggcac aggctcctgc 1884catcagtagc ttcctatgtc atcttcacgt catgcgggtg tttgcatgct gtgctctgga 1944gcttgtcctg tcttctggaa gccctgggcc gggcgtgtga agacttccca gcagtcctat 2004ccacgcacct cagctgaggc cacgggcaca ctgctgcttc ctcactccag ctacgttgtg 2064ttgaacacaa ctgatcctcc aggtgcttgt ggtgcaggaa acgggacgaa cagagcacct 2124gaacccttgc catctgacat caccgacaca ggagaacagt cctctcctct cctctcctct 2184cctctcctag gacagtcccc ggctctggaa tcatgttctt ctcactcatg gtagccagct 2244aagaaagctg caaaccgaac aaagggagaa ccgagctcct gaagccagga gctcctttta 2304ctgtccttct caaaataggg tcattagaca cagccaagtc gtcaaaggcc cctttccttg 2364tacggggccc ccccgccccc ggcagcttga cactgatttc agtgtctatt tggggagaaa 2424gcaattttgt cttggaattt tgtatgttgt aggaatcctt agagagtgtg gttccttctg 2484atggggagaa agggcaaatt attttaatat tttgtatttc acctttataa acatgaatcc 2544tcaggggtga agaacagttt gcataatttt ctgaatttca ggcactttgt gctatatgag 2604gacccatata tttaagcttt ttgtgcagta agaaagtgta aagccaattc cagtgttgga 2664cgaaacaggt ctcgtattta ggtcaaggtg tctccattct ctatcagtgc agggacatgc 2724agtttctgtg gggcagggta ggaccctgca tcatttggag cccagaagga ggccgactgg 2784ccaggcctca ccgcctcagt atgcagtcca gctccacgtc atcccctcac aatggttagt 2844agcaacgtct gggtttgaac gccaggcgtg gttatattat tgaggatgcc tttgcacatg 2904tggccatgct gtgttaggac tgtgccccag ggcccggact tgaagctaga gctggcagaa 2964gagctcctgg catccatggt gcgatgctgc cgccacccag tttctccatt ggaagacaag 3024ggaatgagaa gactgctgtg tatgtgtatt tgtgaacttg gttgtgatct ggtatgccat 3084aggatgtcag acaatatcac tggttaaagt aaagcctatt tttcagat 3132 <210> SEQ IDNO 46 <211> LENGTH: 360 <212> TYPE: PRT <213> ORGANISM: Rattusnorvegicus <400> SEQUENCE: 46 Met Ser Gln Glu Arg Pro Thr Phe Tyr ArgGln Glu Leu Asn Lys Thr 1 5 10 15 Val Trp Glu Val Pro Glu Arg Tyr GlnAsn Leu Ser Pro Val Gly Ser 20 25 30 Gly Ala Tyr Gly Ser Val Cys Ala AlaPhe Asp Thr Lys Thr Gly His 35 40 45 Arg Val Ala Val Lys Lys Leu Ser ArgPro Phe Gln Ser Ile Ile His 50 55 60 Ala Lys Arg Thr Tyr Arg Glu Leu ArgLeu Leu Lys His Met Lys His 65 70 75 80 Glu Asn Val Ile Gly Leu Leu AspVal Phe Thr Pro Ala Arg Ser Leu 85 90 95 Glu Glu Phe Asn Asp Val Tyr LeuVal Thr His Leu Met Gly Ala Asp 100 105 110 Leu Asn Asn Ile Val Lys CysGln Lys Leu Thr Asp Asp His Val Gln 115 120 125 Phe Leu Ile Tyr Gln IleLeu Arg Gly Leu Lys Tyr Ile His Ser Ala 130 135 140 Asp Ile Ile His ArgAsp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu 145 150 155 160 Asp Cys GluLeu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp 165 170 175 Asp GluMet Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu 180 185 190 IleMet Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser 195 200 205Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro 210 215220 Gly Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu Arg Leu Val Gly 225230 235 240 Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser AlaArg 245 250 255 Asn Tyr Ile Gln Ser Leu Ala Gln Met Pro Lys Met Asn PheAla Asn 260 265 270 Val Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu LeuGlu Lys Met 275 280 285 Leu Val Leu Asp Ser Asp Lys Arg Ile Thr Ala AlaGln Ala Leu Ala 290 295 300 His Ala Tyr Phe Ala Gln Tyr His Asp Pro AspAsp Glu Pro Val Ala 305 310 315 320 Glu Pro Tyr Asp Gln Ser Phe Glu SerArg Asp Phe Leu Ile Asp Glu 325 330 335 Trp Lys Ser Leu Thr Tyr Asp GluVal Ile Ser Phe Val Pro Pro Pro 340 345 350 Leu Asp Gln Glu Glu Met GluSer 355 360 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 47 ctgcgacatt ttccagcggc 20 <210> SEQID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: antisense sequence<400> SEQUENCE: 48 ggtaagcttc tgacacttca 20 <210> SEQ ID NO 49 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 49ggccagagac tgaatgtagt 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 50 catcatcagg gtcgtggtac20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 51 ggcacaaagc taatgacttc 20 <210> SEQ ID NO 52<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 52 aggtgctcag gactccattt 20 <210> SEQ ID NO 53 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 53ggatggacag aacagaagca 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 54 gagcaggcag actgccaagg20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 55 aggctagagc ccaggagcca 20 <210> SEQ ID NO 56<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 56 gagcctgtgc ctggcactgg 20 <210> SEQ ID NO 57 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 57tgcaccacaa gcacctggag 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 58 ggctaccatg agtgagaaga20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 59 gtccctgcac tgatagagaa 20 <210> SEQ ID NO 60<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 60 tcttccaatg gagaaactgg 20 <210> SEQ ID NO 61 <211> LENGTH:749 <212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 61tgctgggcgt ggggcgcggg ccgggtgctg cgcgcgggga tccggggcgc tcgctccagc 60tgcttctgtg gatatgtcgg gtccgcgcgc gggattctac cggcaagagc tgaacaaaac 120agtatgggag gtgccgcagc ggctgcaggg cctacgcccg gtgggctccg gcgcctacgg 180ctcagtctgc tcggcctacg acgcgcggct gcgccagaag gtggctgtaa agaagctgtc 240tcgccctttc caatcgctga tccacgcgag gaggacatac cgtgagctgc gcctactcaa 300gcacctgaag cacgagaacg tcataggact tttggacgtc ttcacgccgg ccacatccat 360cgaggatttc agcgaagtgt acctcgtgac gaccctgatg ggcgccgacc tgaataacat 420cgtcaagtgt caggccctga gcgatgagca tgttcaattc cttgtctacc agctgctgcg 480tgggctgaag tatatccact cggcgggcat cattcaccgg gacctgaagc ccagcaatgt 540agcggtgaac gaggactgcg agctgaggat cctggacttt gggctagcac gccaggctga 600tgaggagatg accggatatg tggccacacg gtggtaccgg gcgccagaga tcatgctaaa 660ctggatgcac tacaaccaga cagtggacat ctggtctgtg gcctgcttca tggcttgaac 720tgctggaagg gaagggcctt ctttcctgg 749 <210> SEQ ID NO 62 <400> SEQUENCE:62 000 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 63 cacagaagca gctggagcga 20 <210> SEQID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: antisense sequence<400> SEQUENCE: 64 tgcggcacct cccatactgt 20 <210> SEQ ID NO 65 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 65ccctgcagcc gctgcggcac 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 66 gcagactgag ccgtaggcgc20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 67 ttacagccac cttctggcgc 20 <210> SEQ ID NO 68<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 68 gtatgtcctc ctcgcgtgga 20 <210> SEQ ID NO 69 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 69atggatgtgg ccggcgtgaa 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 70 gaattgaaca tgctcatcgc20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 71 acattgctgg gcttcaggtc 20 <210> SEQ ID NO 72<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 72 atcctcagct cgcagtcctc 20 <210> SEQ ID NO 73 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 73taccaccgtg tggccacata 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 74 cagtttagca tgatctctgg20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 75 caggccacag accagatgtc 20 <210> SEQ ID NO 76<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 76 ccttccagca gttcaagcca 20 <210> SEQ ID NO 77 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: control sequence <400> SEQUENCE: 77 cagcaccatggacgcggaac 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 78 ctgagacatt ttccagcggc 20 <210> SEQID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: antisense sequence<400> SEQUENCE: 79 acgctcgggc acctcccaga 20 <210> SEQ ID NO 80 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 80agcttcttca ctgccacacg 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 81 aatgatggac tgaaatggtc20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 82 tccaacagac caatcacatt 20 <210> SEQ ID NO 83<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 83 tgtaagcttc tgacatttca 20 <210> SEQ ID NO 84 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 84tgaatgtata tactttagac 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 85 ctcacagtct tcattcacag20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 86 cacgtagcct gtcatttcat 20 <210> SEQ ID NO 87<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 87 catcccactg accaaatatc 20 <210> SEQ ID NO 88 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 88tatggtctgt accaggaaac 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 89 agtcaaagac tgaatatagt20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 90 ttctcttatc tgagtccaat 20 <210> SEQ ID NO 91<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 91 catcatcagg atcgtggtac 20 <210> SEQ ID NO 92 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 92tcaaaggact gatcataagg 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 93 ggcacaaagc tgatgacttc20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 94 aggtgctcag gactccatct 20 <210> SEQ ID NO 95<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 95 gcaacaagag gcacttgaat 20 <210> SEQ ID NO 96 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 96ttatcctagc ttagacctat 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 97 acagacggag ccgtaggcgc20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 98 caccgccacc ttctggcgca 20 <210> SEQ ID NO 99<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 99 gtacgttctg cgcgcgtgga 20 <210> SEQ ID NO 100 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 100atggacgtgg ccggcgtgaa 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 101 caggaattgaacgtgctcgt 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 102 acgttgctgg gcttcaggtc 20 <210>SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: antisensesequence <400> SEQUENCE: 103 taccagcgcg tggccacata 20 <210> SEQ ID NO104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: antisense sequence <400>SEQUENCE: 104 cagttgagca tgatctcagg 20 <210> SEQ ID NO 105 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: antisense sequence <400> SEQUENCE: 105cggaccagat atccactgtt 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: antisense sequence <400> SEQUENCE: 106 tgccctggagcagctcagcc 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:antisense sequence <400> SEQUENCE: 107 gttcgatcgg ctcgtgtcga 20 <210>SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 108 gatgagtgga aaagcctgac 20 <210> SEQ ID NO 109 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 109ctgcaacaag aggcacttga 20 <210> SEQ ID NO 110 <211> LENGTH: 50 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Probe <400> SEQUENCE: 110 gatgaagtca tcagctttgtgccaccaccc cttgaccaag aagagatgga 50 <210> SEQ ID NO 111 <211> LENGTH: 19<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: PCR Primer <400> SEQUENCE: 111 gaaggtgaag gtcggagtc19 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 112 gaagatggtg atgggatttc 20 <210> SEQ ID NO 113 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 113caagcttccc gttctcagcc 20 <210> SEQ ID NO 114 <211> LENGTH: 3450 <212>TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (297)...(1379) <400> SEQUENCE: 114 cgggcgctga agcgcgagcgggtgtcttgc ggcgtcggcg tgcgctccct ccccggggag 60 cggctgcagg aggaccgcggcgggagcagc ctcgagccgt gcagccggct ccggcacctt 120 gccgacgctc gtaggagccgccgcggctga caggggcggc gggtcgcagc ctccacacct 180 gcgcgggtgg cgggcgcggggtccggtctg ccgcgggcgg gcgcagagga gagcgtgcgg 240 ctgcaggcag gagcccccgctcggccacct cctcgccccg ctgctgccgc tggaag atg 299 Met 1 tcg cag gag aggccc acg ttc tac cgg cag gag ctg aac aag acc atc 347 Ser Gln Glu Arg ProThr Phe Tyr Arg Gln Glu Leu Asn Lys Thr Ile 5 10 15 tgg gag gtg ccc gaacga tac cag aac ctg tcc ccg gtg ggc tcg ggc 395 Trp Glu Val Pro Glu ArgTyr Gln Asn Leu Ser Pro Val Gly Ser Gly 20 25 30 gcc tat ggc tcg gtg tgtgct gct ttt gat aca aag acg ggg cat cgt 443 Ala Tyr Gly Ser Val Cys AlaAla Phe Asp Thr Lys Thr Gly His Arg 35 40 45 gtg gca gtt aag aag ctg tcgaga ccg ttt cag tcc atc att cac gcc 491 Val Ala Val Lys Lys Leu Ser ArgPro Phe Gln Ser Ile Ile His Ala 50 55 60 65 aaa agg acc tac cga gag ttgcgt ctg ctg aag cac atg aaa cac gaa 539 Lys Arg Thr Tyr Arg Glu Leu ArgLeu Leu Lys His Met Lys His Glu 70 75 80 aat gtg att ggt ctg ttg gat gtgttc aca ccc gca agg tca ctg gag 587 Asn Val Ile Gly Leu Leu Asp Val PheThr Pro Ala Arg Ser Leu Glu 85 90 95 gaa ttc aat gac gtg tac ctg gtg acccat ctc atg ggg gcg gac ctg 635 Glu Phe Asn Asp Val Tyr Leu Val Thr HisLeu Met Gly Ala Asp Leu 100 105 110 aac aac atc gtg aag tgc cag aag ctgacc gac gac cac gtt cag ttt 683 Asn Asn Ile Val Lys Cys Gln Lys Leu ThrAsp Asp His Val Gln Phe 115 120 125 ctc atc tac cag atc ctc cga ggg ctgaag tat ata cat tcg gct gac 731 Leu Ile Tyr Gln Ile Leu Arg Gly Leu LysTyr Ile His Ser Ala Asp 130 135 140 145 ata att cac agg gac cta aag cccagc aac cta gct gtg aac gaa gac 779 Ile Ile His Arg Asp Leu Lys Pro SerAsn Leu Ala Val Asn Glu Asp 150 155 160 tgt gag ctc aag att ctg gat tttggg ctg gct cgg cac act gat gat 827 Cys Glu Leu Lys Ile Leu Asp Phe GlyLeu Ala Arg His Thr Asp Asp 165 170 175 gag atg aca ggc tac gtg gct accagg tgg tac cga gcc cca gag atc 875 Glu Met Thr Gly Tyr Val Ala Thr ArgTrp Tyr Arg Ala Pro Glu Ile 180 185 190 atg ctg aat tgg atg cac tat aaccag aca gtg gat att tgg tcc gtg 923 Met Leu Asn Trp Met His Tyr Asn GlnThr Val Asp Ile Trp Ser Val 195 200 205 ggc tgc atc atg gct gag ctg ttgacc gga aga acg ttg ttt cct ggt 971 Gly Cys Ile Met Ala Glu Leu Leu ThrGly Arg Thr Leu Phe Pro Gly 210 215 220 225 aca gac cat att gat cag ttgaag ctc att tta aga ctc gtt gga acc 1019 Thr Asp His Ile Asp Gln Leu LysLeu Ile Leu Arg Leu Val Gly Thr 230 235 240 cca ggg gct gag ctt ctg aagaaa atc tcc tca gag tct gca aga aac 1067 Pro Gly Ala Glu Leu Leu Lys LysIle Ser Ser Glu Ser Ala Arg Asn 245 250 255 tac att cag tct ctg gcc cagatg ccg aag atg aac ttc gca aat gta 1115 Tyr Ile Gln Ser Leu Ala Gln MetPro Lys Met Asn Phe Ala Asn Val 260 265 270 ttt att ggt gcc aat ccc ctggct gtc gac cta ctg gag aag atg ctc 1163 Phe Ile Gly Ala Asn Pro Leu AlaVal Asp Leu Leu Glu Lys Met Leu 275 280 285 gtt ttg gac tca gat aag aggatc aca gca gcc caa gct ctt gcg cat 1211 Val Leu Asp Ser Asp Lys Arg IleThr Ala Ala Gln Ala Leu Ala His 290 295 300 305 gcc tac ttt gct cag taccac gac cct gat gat gag cct gtt gct gac 1259 Ala Tyr Phe Ala Gln Tyr HisAsp Pro Asp Asp Glu Pro Val Ala Asp 310 315 320 cct tat gac cag tcc tttgaa agc agg gac ctt ctc ata gat gag tgg 1307 Pro Tyr Asp Gln Ser Phe GluSer Arg Asp Leu Leu Ile Asp Glu Trp 325 330 335 aag agc ctg acc tat gatgaa gtc atc agc ttt gtg cca cca ccc ctt 1355 Lys Ser Leu Thr Tyr Asp GluVal Ile Ser Phe Val Pro Pro Pro Leu 340 345 350 gac caa gaa gaa atg gagtcc tga gcacctggtt tctgttctgt ctatctcact 1409 Asp Gln Glu Glu Met GluSer 355 360 tcactgtgag gggaagacct tctcatggga actctccaaa taccattcaagtgcctcttg 1469 ttgaaagatt ccttcatggt ggaagggggt gcatgtatgt gttagtgtttgtgtgtgtgt 1529 gtgtgtctgt ctgttcgtct gtccacctat ctttgtggaa gtcactgtgatggtagtgac 1589 tttatgagtt gtgaatggtc cttggcagtc tgcctgcttt ctcagagtctgggcaggccg 1649 atgggaactg tcatctcctt agggatgtgt gtgttcagtg caaagtaagaaatatgaaaa 1709 tatccctgtt cttagttacc ttgccacttt ggcttctcct gtggccctgcctttaccata 1769 tcagtgacag agagaggctg cttcaggtct gaggctatcc ctcagccatgcataaagtcc 1829 aagagaacca actggctcct ggtctctagc ctgtgaccgg cttgcttaatgtcctcagaa 1889 cctgacaggt atgttcaaaa ctgtcagtct gtttgtgcct taaaagggtgagaagggcgc 1949 gtagatagtt acagagtctc agctgctgac gttctgagcc aggcaagtgcacggggctgt 2009 tggatggcca gtggggagct ggaaaaaaca aggcagcctt taggaaggccatggtgcatg 2069 tgtgtgcatg cgtgtatgtg cagccgccct ccctcacttc aggagcaagctgtttgctgt 2129 gcttaccctt cacctcagtg cagaggtctc cagtgccgag cacaggcacctgccatcagt 2189 agttcctgtg tcatcttcac atctagcaga gcacggatgt gtttgcatgctgtgctcttg 2249 gagcttgtcc tgtcttctgg aagccctgga caaggcgtgt gaaggcttcccagaagttcc 2309 tgtccacatt gcctccgccc accgacgcca tgggcacact gctccctcctcctcctccag 2369 ctactttgtg ttgaacacaa ttgattctcc aggtgctcat ggtgcaggaaaacaggacag 2429 acagagagca ctgaaccctt gccatctgat gtcaccaatt caggaaaacgagtcctctcc 2489 taggactatc cccggttctg gaaatcatgt tctcctcact catggtgacaagctaagaaa 2549 gctgaacaaa gggagagacg agagcgcctg aagccaggag ctcctttactatctttctca 2609 aaagggttgt tagacacaaa ccaagtcatc aaggccccgc tcctctcctcggaagggtcc 2669 cccacccccc ggcagcttga cactgaatcc agtgtcaatt tggggagaaagcagttttgt 2729 cttggaattt tgtatgttgt aggaatcctt agagagtgtg gttccttctgatggggagaa 2789 agggcaaatt attttaatat tttgtatttt cacctttata aacatgaatcctcaggggtg 2849 aagaactgtt tgcataattt tctgaatttt gagcactttg tgctatataaggacccatat 2909 ttaagctttg tgtgcagtaa gaaagtgtaa agccaattcc agtgttggacgtgacaggtc 2969 ttgtgtttag gtcaaggtgt ctcctctcag tgcagggaca tgcctgctctgtggggcagg 3029 cgaggaccct gaatcatttg gagcccagaa ggaggcagac tggccaggtctcaccacctc 3089 agtgtgcagt tcaactccat gccatcccat caagatgggt tagtagcagtgtctgttttt 3149 gaatgccaag tgtgatttcc aacaattctg ctctggttat ttcattgaagacatctttgc 3209 acatgtgacc atgctgtgtt aggggctgtg ttccagggac tggactcgaagctagaactg 3269 gcagaagagt tctggcatcc acagcgcaat gctgccacca cccagtttcttcatcagaag 3329 acaagggaac gagaaaactg ctgttcgttt gtatttgtga acttggctgtaatctggtat 3389 gccataggat gtcagataat accactggtt aaaataaagc ctagttttcaaattcaaccg 3449 g 3450 <210> SEQ ID NO 115 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 115 aagggaacga gaaaactgct gtt 23<210> SEQ ID NO 116 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer<400> SEQUENCE: 116 tattttaacc agtggtatta tctgacatcc t 31 <210> SEQ IDNO 117 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400>SEQUENCE: 117 ttgtatttgt gaacttggct gtaatctggt atgcc 35 <210> SEQ ID NO118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 118ggcaaattca acggcacagt 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 119 gggtctcgct cctggaagat 20<210> SEQ ID NO 120 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 120 aaggccgaga atgggaagct tgtcatc 27 <210> SEQ ID NO 121<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 121atcatttgga gcccagaagg a 21 <210> SEQ ID NO 122 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 122 tggagctgga ctgcatactg a 21<210> SEQ ID NO 123 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 123 ctggccaggc ctcaccgc 18 <210> SEQ ID NO 124 <211>LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 124tgttctagag acagccgcat ctt 23 <210> SEQ ID NO 125 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: PCR Primer <400> SEQUENCE: 125 caccgacctt caccatcttg t 21<210> SEQ ID NO 126 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe<400> SEQUENCE: 126 ttgtgcagtg ccagcctcgt ctca 24 <210> SEQ ID NO 127<211> LENGTH: 3757 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (363)...(1445) <400>SEQUENCE: 127 ggaaccgcga ccactggagc cttagcgggc gcagcagctg gaacgggagtactgcgacgc 60 agcccggagt cggccttgta ggggcgaagg tgcagggaga tcgcggcgggcgcagtcttg 120 agcgccggag cgcgtccctg cccttagcgg ggcttgcccc agtcgcaggggcacatccag 180 ccgctgcggc tgacagcagc cgcgcgcgcg ggagtctgcg gggtcgcggcagccgcacct 240 gcgcgggcga ccagcgcaag gtccccgccc ggctgggcgg gcagcaagggccggggagag 300 ggtgcgggtg caggcggggg ccccacaggg ccaccttctt gcccggcggctgccgctgga 360 aa atg tct cag gag agg ccc acg ttc tac cgg cag gag ctgaac aag 407 Met Ser Gln Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn Lys1 5 10 15 aca atc tgg gag gtg ccc gag cgt tac cag aac ctg tct cca gtgggc 455 Thr Ile Trp Glu Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly20 25 30 tct ggc gcc tat ggc tct gtg tgt gct gct ttt gac aca aaa acg ggg503 Ser Gly Ala Tyr Gly Ser Val Cys Ala Ala Phe Asp Thr Lys Thr Gly 3540 45 tta cgt gtg gca gtg aag aag ctc tcc aga cca ttt cag tcc atc att551 Leu Arg Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile 5055 60 cat gcg aaa aga acc tac aga gaa ctg cgg tta ctt aaa cat atg aaa599 His Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys 6570 75 cat gaa aat gtg att ggt ctg ttg gac gtt ttt aca cct gca agg tct647 His Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser 8085 90 95 ctg gag gaa ttc aat gat gtg tat ctg gtg acc cat ctc atg ggg gca695 Leu Glu Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala 100105 110 gat ctg aac aac att gtg aaa tgt cag aag ctt aca gat gac cat gtt743 Asp Leu Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val 115120 125 cag ttc ctt atc tac caa att ctc cga ggt cta aag tat ata cat tca791 Gln Phe Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser 130135 140 gct gac ata att cac agg gac cta aaa cct agt aat cta gct gtg aat839 Ala Asp Ile Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn 145150 155 gaa gac tgt gag ctg aag att ctg gat ttt gga ctg gct cgg cac aca887 Glu Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr 160165 170 175 gat gat gaa atg aca ggc tac gtg gcc act agg tgg tac agg gctcct 935 Asp Asp Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro180 185 190 gag atc atg ctg aac tgg atg cat tac aac cag aca gtt gat atttgg 983 Glu Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp195 200 205 tca gtg gga tgc ata atg gcc gag ctg ttg act gga aga aca ttgttt 1031 Ser Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe210 215 220 cct ggt aca gac cat att aac cag ctt cag cag att atg cgt ctgaca 1079 Pro Gly Thr Asp His Ile Asn Gln Leu Gln Gln Ile Met Arg Leu Thr225 230 235 gga aca ccc ccc gct tat ctc att aac agg atg cca agc cat gaggca 1127 Gly Thr Pro Pro Ala Tyr Leu Ile Asn Arg Met Pro Ser His Glu Ala240 245 250 255 aga aac tat att cag tct ttg act cag atg ccg aag atg aacttt gcg 1175 Arg Asn Tyr Ile Gln Ser Leu Thr Gln Met Pro Lys Met Asn PheAla 260 265 270 aat gta ttt att ggt gcc aat ccc ctg gct gtc gac ttg ctggag aag 1223 Asn Val Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu Leu GluLys 275 280 285 atg ctt gta ttg gac tca gat aag aga att aca gcg gcc caagcc ctt 1271 Met Leu Val Leu Asp Ser Asp Lys Arg Ile Thr Ala Ala Gln AlaLeu 290 295 300 gca cat gcc tac ttt gct cag tac cac gat cct gat gat gaacca gtg 1319 Ala His Ala Tyr Phe Ala Gln Tyr His Asp Pro Asp Asp Glu ProVal 305 310 315 gcc gat cct tat gat cag tcc ttt gaa agc agg gac ctc cttata gat 1367 Ala Asp Pro Tyr Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu IleAsp 320 325 330 335 gag tgg aaa agc ctg acc tat gat gaa gtc atc agc tttgtg cca cca 1415 Glu Trp Lys Ser Leu Thr Tyr Asp Glu Val Ile Ser Phe ValPro Pro 340 345 350 ccc ctt gac caa gaa gag atg gag tcc tga gcacctggtttctgttctgt 1465 Pro Leu Asp Gln Glu Glu Met Glu Ser 355 360 tgatcccacttcactgtgag gggaaggcct tttcacggga actctccaaa tattattcaa 1525 gtgcctcttgttgcagagat ttcctccatg gtggaagggg gtgtgcgtgc gtgtgcgtgc 1585 gtgttagtgtgtgtgcatgt gtgtgtctgt ctttgtggga gggtaagaca atatgaacaa 1645 actatgatcacagtgacttt acaggaggtt gtggatgctc cagggcagcc tccaccttgc 1705 tcttctttctgagagttggc tcaggcagac aagagctgct gtccttttag gaatatgttc 1765 aatgcaaagtaaaaaaatat gaattgtccc caatcccggt catgcttttg ccactttggc 1825 ttctcctgtgaccccacctt gacggtgggg cgtagacttg acaacatccc acagtggcac 1885 ggagagaaggcccatacctt ctggttgctt cagacctgac accgtccctc agtgatacgt 1945 acagccaaaaaggaccaact ggcttctgtg cactagcctg tgattaactt gcttagtatg 2005 gttctcagatcttgacagta tatttgaaac tgtaaatatg tttgtgcctt aaaaggagag 2065 aagaaagtgtagatagttaa aagactgcag ctgctgaagt tctgagccgg gcaagtcgag 2125 agggctgttggacagctgct tgtgggcccg gagtaatcag gcagccttca taggcggtca 2185 tgtgtgcatgtgagcacatg cgtatatgtg cgtctctctt tctccctcac ccccaggtgt 2245 tgccatttctctgcttaccc ttcacctttg gtgcagaggt ttcttgaata tctgccccag 2305 tagtcagaagcaggttcttg atgtcatgta cttcctgtgt actctttatt tctagcagag 2365 tgaggatgtgttttgcacgt cttgctattt gagcatgcac agctgcttgt cctgctctct 2425 tcaggaggccctggtgtcag gcaggtttgc cagtgaagac ttcttgggta gtttagatcc 2485 catgtcacctcagctgatat tatggcaagt gatatcacct ctcttcagcc cctagtgcta 2545 ttctgtgttgaacacaattg atacttcagg tgcttttgat gtgaaaatca tgaaaagagg 2605 aacaggtggatgtatagcat ttttattcat gccatctgtt ttcaaccaac tatttttgag 2665 gaattatcatgggaaaagac cagggctttt cccaggaata tcccaaactt cggaaacaag 2725 ttattctcttcactcccaat aactaatgct aagaaatgct gaaaatcaaa gtaaaaaatt 2785 aaagcccataaggccagaaa ctccttttgc tgtctttctc taaatatgat tactttaaaa 2845 taaaaaagtaacaaggtgtc ttttccactc ctatggaaaa gggtcttctt ggcagcttaa 2905 cattgacttcttggtttggg gagaaataaa ttttgtttca gaattttgta tattgtagga 2965 atccctttgagaatgtgatt ccttttgatg gggagaaagg gcaaattatt ttaatatttt 3025 gtattttcaactttataaag ataaaatatc ctcaggggtg gagaagtgtc gttttcataa 3085 cttgctgaatttcaggcatt ttgttctaca tgaggactca tatatttaag ccttttgtgt 3145 aataagaaagtataaagtca cttccagtgt tggctgtgtg acagaatctt gtatttgggc 3205 caaggtgtttccatttctca atcagtgcag tgatacatgt actccagagg gacagggtgg 3265 accccctgagtcaactggag caagaaggaa ggaggcagac tgatggcgat tccctctcac 3325 ccgggactctccccctttca aggaaagtga acctttaaag taaaggcctc atctccttta 3385 ttgcagttcaaatcctcacc atccacagca agatgaattt tatcagccat gtttggttgt 3445 aaatgctcgtgtgatttcct acagaaatac tgctctgaat attttgtaat aaaggtcttt 3505 gcacatgtgaccacatacgt gttaggaggc tgcatgctct ggaagcctgg actctaagct 3565 ggagctcttggaagagctct tcggtttctg agcataatgc tcccatctcc tgatttctct 3625 gaacagaaaacaaaagagag aatgagggaa attgctattt tatttgtatt catgaacttg 3685 gctgtaatcagttatgccgt ataggatgtc agacaatacc actggttaaa ataaagccta 3745 tttttcaaattt 3757 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Antisense Oligonucleotide <400> SEQUENCE: 128 gagcaaagta ggcatgtgca 20<210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 129 gtttccgaag tttgggatat 20 <210> SEQID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 130 gcattagtta ttgggagtga 20 <210> SEQID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 131 ccctggagca tccacaacct 20 <210> SEQID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 132 tgtaccagga aacaatgttc 20 <210> SEQID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 133 cgggcaagaa ggtggccctg 20 <210> SEQID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 134 atcgccatca gtctgcctcc 20 <210> SEQID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 135 tgacatcaag aacctgcttc 20 <210> SEQID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 136 ggcccacaag cagctgtcca 20 <210> SEQID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 137 tgaaaacgac acttctccac 20 <210> SEQID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 138 ggtgagaggg aatcgccatc 20 <210> SEQID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 139 atactgtcaa gatctgagaa 20 <210> SEQID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 140 tttccgaagt ttgggatatt 20 <210> SEQID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 141 agagagacgc acatatacgc 20 <210> SEQID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 142 caagaggcac ttgaataata 20 <210> SEQID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 143 attcctccag agaccttgca 20 <210> SEQID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 144 aagacacctt gttacttttt 20 <210> SEQID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 145 tgccctttct ccccatcaaa 20 <210> SEQID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 146 tggcatcctg ttaatgagat 20 <210> SEQID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 147 aaggccttcc cctcacagtg 20 <210> SEQID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 148 aataggcttt attttaacca 20 <210> SEQID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 149 acccaagaag tcttcactgg 20 <210> SEQID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 150 tttcttatta cacaaaaggc 20 <210> SEQID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 151 ggaaatcaca cgagcattta 20 <210> SEQID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 152 ggtccctgtg aattatgtca 20 <210> SEQID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 153 aatatatgag tcctcatgta 20 <210> SEQID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 154 ctaacacgta tgtggtcaca 20 <210> SEQID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 155 tttctcccca tcaaaaggaa 20 <210> SEQID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 156 ctgaacatgg tcatctgtaa 20 <210> SEQID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 157 ataactgatt acagccaagt 20 <210> SEQID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 158 ttctcaaagg gattcctaca 20 <210> SEQID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 159 tctgccccca tgagatgggt 20 <210> SEQID NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 160 ttcgcatgaa tgatggactg 20 <210> SEQID NO 161 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 161 tactgagcaa agtaggcatg 20 <210> SEQID NO 162 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 162 gtccctgctt tcaaaggact 20 <210> SEQID NO 163 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 163 catatgttta agtaaccgca 20 <210> SEQID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 164 cacattctca aagggattcc 20 <210> SEQID NO 165 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 165 ggactccatc tcttcttggt caa 23 <210>SEQ ID NO 166 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 166 gaagtgggat caacagaaca gaaa 24 <210>SEQ ID NO 167 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 167 agcccactgg agacaggttc t 21 <210> SEQID NO 168 <211> LENGTH: 3352 <212> TYPE: DNA <213> ORGANISM: M. musculus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (230)...(1312) <400>SEQUENCE: 168 aggaggaccg cggcgggagc agcctcgagc cgtgcagccg gctccggcaccttgccgacg 60 ctcgtaggag ccgccgcggc tgacaggggc ggcgggtcgc agcctccacacctgcgcggg 120 tggcgggcgc ggggtccggt ctgccgcggg cgggcgcaga ggagagcgtgcggctgcagg 180 caggagcccc cgctcggcca cctcctcgcc ccgctgctgc cgctggaag atgtcg cag 238 Met Ser Gln 1 gag agg ccc acg ttc tac cgg cag gag ctg aacaag acc atc tgg gag 286 Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn LysThr Ile Trp Glu 5 10 15 gtg ccc gaa cga tac cag aac ctg tcc ccg gtg ggctcg ggc gcc tat 334 Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly SerGly Ala Tyr 20 25 30 35 ggc tcg gtg tgt gct gct ttt gat aca aag acg gggcat cgt gtg gca 382 Gly Ser Val Cys Ala Ala Phe Asp Thr Lys Thr Gly HisArg Val Ala 40 45 50 gtt aag aag ctg tcg aga ccg ttt cag tcc atc att cacgcc aaa agg 430 Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile His AlaLys Arg 55 60 65 acc tac cga gag ttg cgt ctg ctg aag cac atg aaa cac gaaaat gtg 478 Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His Glu AsnVal 70 75 80 att ggt ctg ttg gat gtg ttc aca ccc gca agg tca ctg gag gaattc 526 Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu Glu Glu Phe85 90 95 aat gac gtg tac ctg gtg acc cat ctc atg ggg gcg gac ctg aac aac574 Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp Leu Asn Asn 100105 110 115 atc gtg aag tgc cag aag ctg acc gac gac cac gtt cag ttt ctcatc 622 Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln Phe Leu Ile120 125 130 tac cag atc ctc cga ggg ctg aag tat ata cat tcg gct gac ataatt 670 Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asp Ile Ile135 140 145 cac agg gac cta aag ccc agc aac cta gct gtg aac gaa gac tgtgag 718 His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu Asp Cys Glu150 155 160 ctc aag att ctg gat ttt ggg ctg gct cgg cac act gat gat gagatg 766 Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp Asp Glu Met165 170 175 aca ggc tac gtg gct acc agg tgg tac cga gcc cca gag atc atgctg 814 Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu180 185 190 195 aat tgg atg cac tat aac cag aca gtg gat att tgg tcc gtgggc tgc 862 Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser Val GlyCys 200 205 210 atc atg gct gag ctg ttg acc gga aga acg ttg ttt cct ggtaca gac 910 Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro Gly ThrAsp 215 220 225 cat att aac cag ctt cag cag ata atg cgt atg acg ggg acaccc cct 958 His Ile Asn Gln Leu Gln Gln Ile Met Arg Met Thr Gly Thr ProPro 230 235 240 gct tat ctc att aac agg atg cca agc cat gag gca aga aactac att 1006 Ala Tyr Leu Ile Asn Arg Met Pro Ser His Glu Ala Arg Asn TyrIle 245 250 255 cag tct ctg gcc cag atg ccg aag atg aac ttc gca aat gtattt att 1054 Gln Ser Leu Ala Gln Met Pro Lys Met Asn Phe Ala Asn Val PheIle 260 265 270 275 ggt gcc aat ccc ctg gct gtc gac cta ctg gag aag atgctc gtt ttg 1102 Gly Ala Asn Pro Leu Ala Val Asp Leu Leu Glu Lys Met LeuVal Leu 280 285 290 gac tca gat aag agg atc aca gca gcc caa gct ctt gcgcat gcc tac 1150 Asp Ser Asp Lys Arg Ile Thr Ala Ala Gln Ala Leu Ala HisAla Tyr 295 300 305 ttt gct cag tac cac gac cct gat gat gag cct gtt gctgac cct tat 1198 Phe Ala Gln Tyr His Asp Pro Asp Asp Glu Pro Val Ala AspPro Tyr 310 315 320 gac cag tcc ttt gaa agc agg gac ctt ctc ata gat gagtgg aag agc 1246 Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu TrpLys Ser 325 330 335 ctg acc tat gat gaa gtc atc agc ttt gtg cca cca cccctt gac caa 1294 Leu Thr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro LeuAsp Gln 340 345 350 355 gaa gaa atg gag tcc tga gcacctggtt tctgttctgtctatctcact 1342 Glu Glu Met Glu Ser 360 tcactgtgag gggaagacct tctcatgggaactctccaaa taccattcaa gtgcctcttg 1402 ttgaaagatt ccttcatggt ggaagggggtgcatgtatgt gttagtgttt gtgtgtgtgt 1462 gtgtgtctgt ctgttcgtct gtccacctatctttgtggaa gtcactgtga tggtagtgac 1522 tttatgagtt gtgaatggtc cttggcagtctgcctgcttt ctcagagtct gggcaggccg 1582 atgggaactg tcatctcctt agggatgtgtgtgttcagtg caaagtaaga aatatgaaaa 1642 tatccctgtt cttagttacc ttgccactttggcttctcct gtggccctgc ctttaccata 1702 tcagtgacag agagaggctg cttcaggtctgaggctatcc ctcagccatg cataaagtcc 1762 aagagaacca actggctcct ggtctctagcctgtgaccgg cttgcttaat gtcctcagaa 1822 cctgacaggt atgttcaaaa ctgtcagtctgtttgtgcct taaaagggtg agaagggcgc 1882 gtagatagtt acagagtctc agctgctgacgttctgagcc aggcaagtgc acggggctgt 1942 tggatggcca gtggggagct ggaaaaaacaaggcagcctt taggaaggcc atggtgcatg 2002 tgtgtgcatg cgtgtatgtg cagccgccctccctcacttc aggagcaagc tgtttgctgt 2062 gcttaccctt cacctcagtg cagaggtctccagtgccgag cacaggcacc tgccatcagt 2122 agttcctgtg tcatcttcac atctagcagagcacggatgt gtttgcatgc tgtgctcttg 2182 gagcttgtcc tgtcttctgg aagccctggacaaggcgtgt gaaggcttcc cagaagttcc 2242 tgtccacatt gcctccgccc accgacgccatgggcacact gctccctcct cctcctccag 2302 ctactttgtg ttgaacacaa ttgattctccaggtgctcat ggtgcaggaa aacaggacag 2362 acagagagca ctgaaccctt gccatctgatgtcaccaatt caggaaaacg agtcctctcc 2422 taggactatc cccggttctg gaaatcatgttctcctcact catggtgaca agctaagaaa 2482 gctgaacaaa gggagagacg agagcgcctgaagccaggag ctcctttact atctttctca 2542 aaagggttgt tagacacaaa ccaagtcatcaaggccccgc tcctctcctc ggaagggtcc 2602 cccacccccc ggcagcttga cactgaatccagtgtcaatt tggggagaaa gcagttttgt 2662 cttggaattt tgtatgttgt aggaatccttagagagtgtg gttccttctg atggggagaa 2722 agggcaaatt attttaatat tttgtattttcacctttata aacatgaatc ctcaggggtg 2782 aagaactgtt tgcataattt tctgaattttgagcactttg tgctatataa ggacccatat 2842 ttaagctttg tgtgcagtaa gaaagtgtaaagccaattcc agtgttggac gtgacaggtc 2902 ttgtgtttag gtcaaggtgt ctcctctcagtgcagggaca tgcctgctct gtggggcagg 2962 cgaggaccct gaatcatttg gagcccagaaggaggcagac tggccaggtc tcaccacctc 3022 agtgtgcagt tcaactccat gccatcccatcaagatgggt tagtagcagt gtctgttttt 3082 gaatgccaag tgtgatttcc aacaattctgctctggttat ttcattgaag acatctttgc 3142 acatgtgacc atgctgtgtt aggggctgtgttccagggac tggactcgaa gctagaactg 3202 gcagaagagt tctggcatcc acagcgcaatgctgccacca cccagtttct tcatcagaag 3262 acaagggaac gagaaaactg ctgttcgtttgtatttgtga acttggctgt aatctggtat 3322 gccataggat gtcagataat accactggtt3352 <210> SEQ ID NO 169 <211> LENGTH: 503 <212> TYPE: DNA <213>ORGANISM: M. musculus <400> SEQUENCE: 169 cgcaagaata aagtcagtggtcacaaatag agggggtcag tggctagaag aagagtaagc 60 ctgaattgag catcccagacagtggtccat acgggccgtc agctagctca ttccctgaga 120 tcactaacac tactgaacatagtcattctg aaagtctgtg tttttacagg caagaaacta 180 cattcagtct ctggcccag atgccg aag atg aac ttc gca aat gta ttt att 232 ggt gcc aat ccc ctg gct gtcgac cta ctg gag aag atg ctc gtt ttg 280 gac tca gat aag agg atc aca gcagcc caa gct ctt gcg cat gct act 328 ttg ctc agt acc acg acc ctg atg atgagc ctg ttg ctg acc ctt atg 376 acc agt cct ttg aaa gca ggg acc ttc tcatag atgagtggaa gagcctgacc 429 tatgatgaag tcatcagctt tgtgccaccaccccttgacc aagaagagat ggagtcctga 489 gcacctggtt tctg 503 <210> SEQ ID NO170 <211> LENGTH: 1500 <212> TYPE: DNA <213> ORGANISM: M. musculus <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (297)...(1073) <400>SEQUENCE: 170 cgggcgctga agcgcgagcg ggtgtcttgc ggcgtcggcg tgcgctccctccccggggag 60 cggctgcagg aggaccgcgg cgggagcagc ctcgagccgt gcagccggctccggcacctt 120 gccgacgctc gtaggagccg ccgcggctga caggggcggc gggtcgcagcctccacacct 180 gcgcgggtgg cgggcgcggg gtccggtctg ccgcgggcgg gcgcagaggagagcgtgcgg 240 ctgcaggcag gagcccccgc tcggccacct cctcgccccg ctgctgccgctggaag atg 299 Met 1 tcg cag gag agg ccc acg ttc tac cgg cag gag ctg aacaag acc atc 347 Ser Gln Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn LysThr Ile 5 10 15 tgg gag gtg ccc gaa cga tac cag aac ctg tcc ccg gtg ggctcg ggc 395 Trp Glu Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly SerGly 20 25 30 gcc tat ggc tcg gtg tgt gct gct ttt gat aca aag acg ggg catcgt 443 Ala Tyr Gly Ser Val Cys Ala Ala Phe Asp Thr Lys Thr Gly His Arg35 40 45 gtg gca gtt aag aag ctg tcg aga ccg ttt cag tcc atc att cac gcc491 Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile His Ala 5055 60 65 aaa agg acc tac cga gag ttg cgt ctg ctg aag cac atg aaa cac gaa539 Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His Glu 7075 80 aat gtg att ggt ctg ttg gat gtg ttc aca ccc gca agg tca ctg gag587 Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu Glu 8590 95 gaa ttc aat gac gtg tac ctg gtg acc cat ctc atg ggg gcg gac ctg635 Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp Leu 100105 110 aac aac atc gtg aag tgc cag aag ctg acc gac gac cac gtt cag ttt683 Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln Phe 115120 125 ctc atc tac cag atc ctc cga ggg ctg aag tat ata cat tcg gct gac731 Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asp 130135 140 145 ata att cac agg gac cta aag ccc agc aac cta gct gtg aac gaagac 779 Ile Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu Asp150 155 160 tgt gag ctc aag att ctg gat ttt ggg ctg gct cgg cac act gatgat 827 Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp Asp165 170 175 gag atg aca ggc tac gtg gct acc agg tgg tac cga gcc cca gagatc 875 Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile180 185 190 atg ctg aat tgg atg cac tat aac cag aca gtg gat att tgg tccgtg 923 Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser Val195 200 205 ggc tgc atc atg gct gag ctg ttg acc gga aga acg ttg ttt cctggt 971 Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro Gly210 215 220 225 aca gac cat att gat cag ttg aag ctc att tta aga ctc gttgga acc 1019 Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu Arg Leu Val GlyThr 230 235 240 cca ggg gct gag ctt ctg aag aaa atc tcc tca gag tct gatgcc aag 1067 Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser Asp AlaLys 245 250 255 cca tga ggtgagaaca aacagcatgc acagggaagt ctacctcggaggccaccttc 1123 Pro tcgtggtagt gtctgtgtat agccagcagt ttctaatgtcaccgaatgct tgcatgtgcc 1183 ccaagaaccg ttaaagcagt actggctgtg tgctagcggagtgttggcat ttaggatgca 1243 gtctcctgag cctgcgaggc agcgatgcag tgtagggcagtgttccctag tgtttggctt 1303 tctgatcttg tgcttgaggt aacaagtgtc gttgcagttgtatgtagtta gggtgtgcta 1363 cagccgtgtc atgggtgcat ggaacagagt tcattagtgtgctttgctct ccacccattt 1423 tacaaccaag agaagactgc atgcaagcac gcactataaaattccttgtg ctaataaaaa 1483 aaaaaaaaaa aaaaaaa 1500 <210> SEQ ID NO 171<211> LENGTH: 384 <212> TYPE: DNA <213> ORGANISM: M. musculus <400>SEQUENCE: 171 ttgcaaggac gctccagctc gccgcttagt cacataccac tgctcatttcagtattgttt 60 gacaaaacag ttttccatac cgagcagagg ggcgcccctc aagatcaagaagtgctgctt 120 ttgatacaaa gacggggcat cgtgtggcag ttaagaagct gtcgagaccgtttcagtcca 180 tcattcacgc caaaaggacc taccgagagt tgcgtctgct gaagcacatgaaacacgaaa 240 atgtgattgg tctgttggat gtgttcacac ccgcaaggtc actggaggaattcaatgacg 300 tgtacctggt gacccatctc atgggggcgg acctgaacaa catcgtgaagtgccagaagc 360 tgaccgacga ccacgttcag tttc 384 <210> SEQ ID NO 172 <211>LENGTH: 463 <212> TYPE: DNA <213> ORGANISM: M. musculus <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: 429 <223> OTHERINFORMATION: n = A, T, C, or G <400> SEQUENCE: 172 atattgggta agatctggattcagagcggg gcctccttgg agctgttctc gcgagagttc 60 cgcgagaggc tcccggccgctgcctgtggg atcgccgcca ctggagccca agcggggcgc 120 tgaagcgcga gcgggtgtcttgcggcgtcg gcgtgcgctc cctccccggg gagcggctgc 180 aggaggaccg cggcgggagcagcctcgagc cgtgcagccg gctccggcac cttgccgacg 240 ctcgtaggag ccgccgcggctgacaggggc ggcgggtcgc accctccaca cctgcgcggg 300 tggcgggcgc ggggtccggtctgccgcggg cgggcgcaga ggagagcgtg cggctgcagg 360 caggagcccc cgctcggccacctcctcgcc ccgctgctgc cgctggaaga tgtcgcagga 420 gaggcccang ttctaccggcaggagctgaa caagaccatc tgg 463 <210> SEQ ID NO 173 <211> LENGTH: 1083<212> TYPE: DNA <213> ORGANISM: R. norvegicus <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)...(1083) <400> SEQUENCE: 173 atg tctcag gag agg ccc acg ttc tac cgg cag gag ctg aac aag acc 48 Met Ser GlnGlu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn Lys Thr 1 5 10 15 gtc tgggag gtg ccc gag cga tac cag aac ctg tcc ccg gtg ggc tcg 96 Val Trp GluVal Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly Ser 20 25 30 gga gcc tacggc tcg gtg tgt gct gct ttt gat aca aag acg gga cat 144 Gly Ala Tyr GlySer Val Cys Ala Ala Phe Asp Thr Lys Thr Gly His 35 40 45 cgt gtg gca gtgaag aag ctg tcg aga ccg gtt cag ccc atc att cac 192 Arg Val Ala Val LysLys Leu Ser Arg Pro Val Gln Pro Ile Ile His 50 55 60 gcc aaa agg tcc tacagg gag ctg cgg ctg ctg aag cac atg aag cac 240 Ala Lys Arg Ser Tyr ArgGlu Leu Arg Leu Leu Lys His Met Lys His 65 70 75 80 gag aat gtg att ggtctg ttg gat gtg ttt aca cct gca agg tcc ctg 288 Glu Asn Val Ile Gly LeuLeu Asp Val Phe Thr Pro Ala Arg Ser Leu 85 90 95 gag gaa ttc aac gat gtgtac ctg gtg acc cat ctc atg ggg gca gac 336 Glu Glu Phe Asn Asp Val TyrLeu Val Thr His Leu Met Gly Ala Asp 100 105 110 ctg aac aac atc gtg aagtgt cag aag ctt acc gat gac cac gtt cag 384 Leu Asn Asn Ile Val Lys CysGln Lys Leu Thr Asp Asp His Val Gln 115 120 125 ttt ctt atc tac cag atcctg cga ggg ctg aag tat ata cac tcg gct 432 Phe Leu Ile Tyr Gln Ile LeuArg Gly Leu Lys Tyr Ile His Ser Ala 130 135 140 gac ata atc cac agg gaccta aag ccc agc aac ctc gct gtg aat gaa 480 Asp Ile Ile His Arg Asp LeuLys Pro Ser Asn Leu Ala Val Asn Glu 145 150 155 160 gac tgt gag ctg aagatt ctg gat ttt ggg ctg gct cgg cac act gat 528 Asp Cys Glu Leu Lys IleLeu Asp Phe Gly Leu Ala Arg His Thr Asp 165 170 175 gac gaa atg acc ggctac gtg gct acc cgg tgg tac aga gcc ccc gag 576 Asp Glu Met Thr Gly TyrVal Ala Thr Arg Trp Tyr Arg Ala Pro Glu 180 185 190 att atg ctg aat tggatg cac tac aac cag aca gtg gat att tgg tcc 624 Ile Met Leu Asn Trp MetHis Tyr Asn Gln Thr Val Asp Ile Trp Ser 195 200 205 gtg ggc tgc atc atggct gag ctg ttg acc gga aga acg ttg ttt cct 672 Val Gly Cys Ile Met AlaGlu Leu Leu Thr Gly Arg Thr Leu Phe Pro 210 215 220 ggt aca gac cat attgat cag ttg aag ctc att tta aga ctc gtt gga 720 Gly Thr Asp His Ile AspGln Leu Lys Leu Ile Leu Arg Leu Val Gly 225 230 235 240 acc cca ggg gctgag ctt ctg aag aaa atc tcc tca gag tct gca aga 768 Thr Pro Gly Ala GluLeu Leu Lys Lys Ile Ser Ser Glu Ser Ala Arg 245 250 255 aac tac att cagtct ctg gcc cag atg ccg aag atg aac ttc gca aat 816 Asn Tyr Ile Gln SerLeu Ala Gln Met Pro Lys Met Asn Phe Ala Asn 260 265 270 gta ttt att ggtgcc aat ccc ctg gct gtc gac ctg ctg gaa aag atg 864 Val Phe Ile Gly AlaAsn Pro Leu Ala Val Asp Leu Leu Glu Lys Met 275 280 285 ctg gtt ttg gactca gat aag agg atc aca gca gcc caa gct ctt gcg 912 Leu Val Leu Asp SerAsp Lys Arg Ile Thr Ala Ala Gln Ala Leu Ala 290 295 300 cat gcc tac tttgct cag tac cac gac cct gat gat gag cca gtg gct 960 His Ala Tyr Phe AlaGln Tyr His Asp Pro Asp Asp Glu Pro Val Ala 305 310 315 320 gac cct tatgac cag tcc ttt gaa agc agg gac ctc ctt ata gac gaa 1008 Asp Pro Tyr AspGln Ser Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu 325 330 335 tgg aag agcctg acc tac gat gaa gtc att agc ttt gtg cca ccg ccc 1056 Trp Lys Ser LeuThr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro 340 345 350 ctt gac caagaa gaa atg gac tcc tga 1083 Leu Asp Gln Glu Glu Met Asp Ser 355 360<210> SEQ ID NO 174 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 174 gtgcgcgcga gcccgaaatc 20 <210> SEQID NO 175 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 175 ctgcgacatt ttccagcggc 20 <210> SEQID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 176 catcatcagg gtcgtggtac 20 <210> SEQID NO 177 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 177 aggtgctcag gactccattt 20 <210> SEQID NO 178 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 178 gtccctgctt tcaaaggact 20 <210> SEQID NO 179 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 179 ggccagagac tgaatgtagt 20 <210> SEQID NO 180 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 180 agctcctgcc ggtagaacgt 20 <210> SEQID NO 181 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 181 tcaaaagcag cacacaccga 20 <210> SEQID NO 182 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 182 cccgtctttg tatcaaaagc 20 <210> SEQID NO 183 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 183 aacggtctcg acagcttctt 20 <210> SEQID NO 184 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 184 taggtccttt tggcgtgaat 20 <210> SEQID NO 185 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 185 agatgggtca ccaggtacac 20 <210> SEQID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 186 gcccccatga gatgggtcac 20 <210> SEQID NO 187 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 187 tcatcagtgt gccgagccag 20 <210> SEQID NO 188 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 188 gtcaacagct cagccatgat 20 <210> SEQID NO 189 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 189 cgttcttccg gtcaacagct 20 <210> SEQID NO 190 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 190 atcaatatgg tctgtaccag 20 <210> SEQID NO 191 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 191 cttaaaatga gcttcaactg 20 <210> SEQID NO 192 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 192 gggttccaac gagtcttaaa 20 <210> SEQID NO 193 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 193 tcagaagctc agcccctggg 20 <210> SEQID NO 194 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 194 ggagattttc ttcagaagct 20 <210> SEQID NO 195 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 195 cagactctga ggagattttc 20 <210> SEQID NO 196 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 196 tagtttcttg cagactctga 20 <210> SEQID NO 197 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 197 agactgaatg tagtttcttg 20 <210> SEQID NO 198 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 198 ttcatcttcg gcatctgggc 20 <210> SEQID NO 199 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 199 atttgcgaag ttcatcttcg 20 <210> SEQID NO 200 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 200 caataaatac atttgcgaag 20 <210> SEQID NO 201 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 201 ggattggcac caataaatac 20 <210> SEQID NO 202 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 202 gctgctgtga tcctcttatc 20 <210> SEQID NO 203 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 203 aggcatgcgc aagagcttgg 20 <210> SEQID NO 204 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 204 tgagcaaagt aggcatgcgc 20 <210> SEQID NO 205 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 205 tcaaaggact ggtcataagg 20 <210> SEQID NO 206 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 206 catttcttct tggtcaaggg 20 <210> SEQID NO 207 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 207 aggactccat ttcttcttgg 20 <210> SEQID NO 208 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 208 cttcccctca cagtgaagtg 20 <210> SEQID NO 209 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 209 tatttggaga gttcccatga 20 <210> SEQID NO 210 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 210 acttgaatgg tatttggaga 20 <210> SEQID NO 211 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 211 aacaagaggc acttgaatgg 20 <210> SEQID NO 212 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 212 acccccttcc accatgaagg 20 <210> SEQID NO 213 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 213 agcaggcaga ctgccaagga 20 <210> SEQID NO 214 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 214 cacacacatc cctaaggaga 20 <210> SEQID NO 215 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 215 taaaggcagg gccacaggag 20 <210> SEQID NO 216 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 216 gcagcctctc tctgtcactg 20 <210> SEQID NO 217 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 217 gggatagcct cagacctgaa 20 <210> SEQID NO 218 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 218 gcatggctga gggatagcct 20 <210> SEQID NO 219 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 219 gagccagttg gttctcttgg 20 <210> SEQID NO 220 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 220 aggcacaaac agactgacag 20 <210> SEQID NO 221 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 221 ccttttaagg cacaaacaga 20 <210> SEQID NO 222 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 222 gacctctgca ctgaggtgaa 20 <210> SEQID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 223 ggcactggag acctctgcac 20 <210> SEQID NO 224 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 224 agagcacagc atgcaaacac 20 <210> SEQID NO 225 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 225 ccagggcttc cagaagacag 20 <210> SEQID NO 226 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 226 aaggagctcc tggcttcagg 20 <210> SEQID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 227 ggattcctac aacatacaaa 20 <210> SEQID NO 228 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 228 gaaggaacca cactctctaa 20 <210> SEQID NO 229 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 229 tttgcccttt ctccccatca 20 <210> SEQID NO 230 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 230 aatattaaaa taatttgccc 20 <210> SEQID NO 231 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 231 tcatgtttat aaaggtgaaa 20 <210> SEQID NO 232 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 232 ccctgaggat tcatgtttat 20 <210> SEQID NO 233 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 233 ggaattggct ttacactttc 20 <210> SEQID NO 234 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 234 cgtccaacac tggaattggc 20 <210> SEQID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 235 ccttctgggc tccaaatgat 20 <210> SEQID NO 236 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 236 tctgacatcc tatggcatac 20 <210> SEQID NO 237 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 237 gttaatatgg tctgtaccag 20 <210> SEQID NO 238 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 238 gctgaagctg gttaatatgg 20 <210> SEQID NO 239 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 239 cgcattatct gctgaagctg 20 <210> SEQID NO 240 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 240 tgttaatgag ataagcaggg 20 <210> SEQID NO 241 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 241 cttggcatcc tgttaatgag 20 <210> SEQID NO 242 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 242 tgcctcatgg cttggcatcc 20 <210> SEQID NO 243 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 243 actgaatgta gtttcttgcc 20 <210> SEQID NO 244 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 244 cttgcctgta aaaacacaga 20 <210> SEQID NO 245 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 245 tcacctcatg gcttggcatc 20 <210> SEQID NO 246 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 246 tttgttctca cctcatggct 20 <210> SEQID NO 247 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 247 tgctggctat acacagacac 20 <210> SEQID NO 248 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 248 tggaaaactg ttttgtcaaa 20 <210> SEQID NO 249 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 249 actctcgcga gaacagctcc 20 <210> SEQID NO 250 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 250 tcccacaggc agcggccggg 20 <210> SEQID NO 251 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 251 cccgcttggg ctccagtggc 20 <210> SEQID NO 252 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 252 gcagttttct cgttcccttg 20 <210> SEQID NO 253 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 253 ctgagcaaag taggcatgcg 20 <210> SEQID NO 254 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 254 ggaggcaatg tggacaggaa 20 <210> SEQID NO 255 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 255 cattttcgtg tttcatgtgc ttc 23 <210>SEQ ID NO 256 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 256 tattttaacc agtggtatta tctgacatcc t31 <210> SEQ ID NO 257 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 257 ctgcgacatc ttccagcggc 20 <210> SEQID NO 258 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 258 ggtcagcttc tggcacttca 20 <210> SEQID NO 259 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 259 aagcaggcag actgccaagg 20 <210> SEQID NO 260 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 260 aggcatgcgc aagagctt 18 <210> SEQ IDNO 261 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 261 gggacaggtt ctggtatcgc 20 <210> SEQID NO 262 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 262 tctcgtgctt catgtgcttc a 21 <210> SEQID NO 263 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 263 tggagctgga ctgcatactg a 21 <210> SEQID NO 264 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 264 catcagggtc gtggtac 17 <210> SEQ IDNO 265 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: AntisenseOligonucleotide <400> SEQUENCE: 265 catcatcagg gtcgt 15 <210> SEQ ID NO266 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400>SEQUENCE: 266 agctgatctg gcctacagtt 20

What is claimed is:
 1. A method for treating airway hyperresponsivenessor pulmonary inflammation in an individual in need thereof, comprisingadministering to said individual an antisense compound 8 to 30nucleobases in length targeted to a nucleic acid molecule encoding ahuman p38α MAP kinase protein to said individual.
 2. The method of claim1, wherein said antisense compound is an antisense oligonucleotide. 3.The method of claim 2, wherein at least one covalent linkage of saidantisense compound is a modified covalent linkage.
 4. The method ofclaim 2, wherein at least one nucleotide of said antisense compound hasa modified sugar moiety.
 5. The method of claim 2, wherein at least onenucleotide of said antisense compound has a modified nucleobase.
 6. Themethod of claim 1, further comprising administering an anti-asthmamedication to said individual.
 7. The method of claim 1 wherein saidantisense compound comprises at least one lipophilic moiety whichenhances the cellular uptake of said antisense compound.
 8. The methodof claim 1, wherein said antisense compound is aerosolized and inhaledby said individual.
 9. The method of claim 1, wherein said antisensecompound is administered intranasally, intrapulmonarily orintratracheally.
 10. The method of claim 1, wherein said airwayhyperresponsiveness or pulmonary inflammation is associated with asthma.11. A pharmaceutical composition comprising an antisense oligonucleotidetargeted to nucleic acid encoding human p38α MAP kinase in a formulationsuitable for intranasal, intrapulmonary or intratracheal administration.12. The pharmaceutical composition of claim 11, wherein said compositionis in a metered dose inhaler or nebulizer.